Immunoglobulin Heavy Variable Gene Research Articles

Chronic Lymphocytic Leukemia (CLL) with Borderline Immunoglobulin Heavy Chain Mutational Status, a Rare Subgroup of CLL with Variable Disease Course.

Chronic lymphocytic leukemia (CLL) exhibits substantial variability in disease course. The mutational status of the B-cell receptor immunoglobulin heavy variable (IGHV) chain is a critical prognostic factor, categorizing patients into mutated (M-IGHV) and unmutated (U-IGHV) groups. Recently, a third subgroup with borderline mutational status (BL-IGHV) has been identified, comprising approximately 5% of CLL cases. This study retrospectively analyzes the outcomes of 30 BL-IGHV mutated patients among a cohort of 653 CLL patients, focusing on time to first treatment (TTFT) and overall survival (OS). BL-IGHV patients had a short TTFT similar to U-IGHV patients (median 30.2 vs. 34 months; p = 0.9). Conversely, the OS of BL-IGHV patients resembled M-IGHV patients (median NR vs. 258 months; p = 1). Despite a similar incidence in unfavorable prognostic factors, the TTFT was shorter compared to other published cohorts. However, striking similarities with other experiences suggest that BL-IGHV mutated patients share common biological characteristics, biased IGHV gene usage and BCR subset frequency. These findings also underscore the need for multicentric efforts aggregating data on BL-IGHV CLL in order to elucidate its disease course and optimize therapeutic approaches for this rare subgroup. Until then, predicting outcomes and optimal management of BL-IGHV CLL will remain challenging.

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

Precision Targeting of the Malignant Clone in Diffuse Large B Cell Lymphoma Using Chimeric Antigen Receptor T Cells Against the Clonotypic IGHV4-34 B Cell Receptor

Introduction: CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has revolutionized the treatment of B cell lymphoma. However, most CART19-treated patients either fail to respond or show disease progression after an initial response. In fact, up to 30% of lymphoma relapses following CART19 show loss of CD19 expression. In addition, several patients who respond well to CART19 display complete loss or severe reduction of their normal B cell repertoire, leaving them exposed to recurrent infections, limited response to vaccines, and requiring prophylactic measures such as intravenous immunoglobulins. A significant portion of mature B cell malignancies express B cell receptors (BCR) that use the same immunoglobulin heavy variable gene: IGHV4-34. In particular, ~30% of activated B cell (ABC) diffuse large B cell lymphomas (DLBCL), ~35% of primary central nervous system lymphomas, ~65% of vitreoretinal lymphomas, and ~35% of hairy cell leukemia variant express IGHV4-34. This suggests that the IGHV4-34 heavy chain is critical for driving the disease by delivering cell survival and proliferation signals, and multiple studies have shown that BCR signaling is required for ABC-DLBCL survival. However, while highly enriched in several types of B cell lymphomas, IGHV4-34 expressing B cells compose only ~5% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV4-34 CAR T cells would be highly effective and safe against B cell malignancies, as they would: (i) efficiently recognize IGHV4-34+ lymphoma cells while sparing normal B cells; and (ii) target a tumor driver that is essential for lymphoma cell survival ( Fig 1A). Methods and Results:We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV4-34+ BCR (CART4-34) using a single-chain variable fragment (scFv) derived from the 9G4 rat monoclonal antibody. We used tumor B cells that endogenously (HBL1) or exogenously (Jeko1, Bonna12, Mec1) express the IGHV4-34 BCR. Using cytotoxicity, cytokine secretion and proliferation assays in vitro we showed that CART4-34 specifically target all IGHV4-34+ tumor B cells tested while sparing IGHV4-34- tumor B cells and, most importantly, healthy B cells. However, while the initial CART4-34 showed strong cytotoxicity towards IGHV4-34+ cells in short-term in vitro assays, they were significantly inferior to CART19 in in vivo xenograft models. We hypothesized that the poor performance of CART4-34 compared to CART19 was due to the unique challenge of targeting the membrane-distal portion of the BCR, 18 nm from the plasma membrane. In contrast, CART19 targets an epitope of CD19 located just 5 nm from the membrane ( Fig 1B). Therefore, to improve immune synapse (IS) formation, we designed new CAR constructs with smaller extracellular domains by replacing the original CD8 hinge (44 amino acids (aa)) with either: (i) a short version of the IgG4 hinge (12aa); or (ii) a G4S linker (5aa). We found that both CART4-34 (IgG4) and CART4-34 (G4S) exhibited significantly improved cytotoxicity towards IGHV4-34+ tumor cells in short-term and long-term in vitro assays in all IGHV4-34+ cell lines tested, while maintaining specificity. Importantly, in in vivo human xenograft models of IGHV4-34+ lymphoma (HBL1), CART4-34 with short hinges showed significantly improved tumor control comparable to, or better than, CART19 ( Fig 1B). We also found that while CART19-resistant lymphoma cells had complete loss of CD19 surface expression, CART4-34-resistant tumor cells retained high expression of IgM, suggesting a reduced likelihood of antigen-negative escape owing to the critical role of the BCR for lymphoma B cell survival. Mechanistically, we found that while the original CART4-34 (CD8 hinge) formed IS with striking morphological differences compared to IS formed by CART19, the IS formed by CART4-34 with short hinges highly resembled those formed by CART19, providing a rationale for the improved anti-tumor potency of short-hinge CART4-34. Conclusion:We implemented a novel paradigm for the treatment of B cell malignancies: the specific targeting of the malignant clone, while sparing the remainder of the healthy B cell repertoire. We designed and optimized a best-in-class anti-IGHV4-34 CAR T cell product with potent anti-tumor effects and minimal toxicity towards IGHV4-34-negative B cells. The activity of CART4-34 will be investigated in a Phase I clinical trial.

Evidence of Defective Cell-Autonomous Signalling By B Cell Receptors in CLL Stereotyped Subset #1

B cell receptor (BCR)-mediated signalling is central in the pathogenesis of chronic lymphocytic leukaemia (CLL), and represents a target for pharmacological intervention. Notably, so far characterized CLL-derived BCRs are capable of triggering cell-autonomous intracellular signalling through homotypic interactions without requirement for exogenous antigens. Approximately 41% of all CLL cases can be classified in subsets based on homologous, otherwise known as stereotyped, BCRs expressed by the malignant clones, sharing remarkably similar sequences in the VH CDR3 region and highly restricted immunoglobulin heavy and/or light variable genes. Since cases within a subset display clinicobiological consistency in contrast with the pronounced heterogeneity of CLL at large, analysis of the mechanism of BCR-mediated signalling can unveil correlations between molecular details of BCR activation and the course of the disease. Our present study focused on BCRs of stereotyped subset #1, the second largest stereotyped subset, known for a particularly aggressive clinical course. Cases belonging to subset #1 express unmutated IGHV genes of phylogenetic clan I (namely, IGHV1, IGHV5 and IGHV7 subgroup genes) partnered by light chains encoded by the IGKV1-39/1D-39 gene. First, we used X-ray crystallography to determine the high-resolution crystal structures of the BCR Fab fragments from three subset #1 cases utilizing the IGHV1-2, IGHV1-3, and IGHV5-10 genes partnered with IGKV1-39/1D-39 to obtain information on the combining sites' structures and possible modes of intermolecular interactions that may mediate cell-autonomous signalling. Analysis of the experimental structures revealed that, despite the high sequence homology of the three proteins, the heavy and light chain CDR loops in the three BCR Fabs are arranged differently to shape binding cavities with distinct dimensions and polarity, thus suggesting that distinct antigens may be the physiological target of each subgroup. Unlike BCRs from other subsets previously characterised, the intermolecular contacts between the subset #1 Fab molecules in the three crystals involve diverse residues that are not indicative of a shared homotypic interaction. In addition, the surfaces involved in the contacts are small and the computed energies for the protein-protein contacts observed are suggestive at best of very weak interactions. In accordance to this, when we used analytical ultracentrifugation along with chromatographic and calorimetric methods to detect BCR Fab oligomerization in solution, no evidence of self-association was obtained, thus indicating that the receptors in solution either do not interact, or are characterized by self-dissociation constants that exceed the sensitivity limit of the techniques employed. To assess whether the cellular environment could promote self-association and signalling activity of the full-length BCR, we expressed subset #1 BCRs in the model triple knockout (TKO) pre-B-cell line, designed to measure Ca 2+ ion influx using cytofluorimetry. All subset #1 BCRs tested were unable to mobilize autonomously the Ca 2+ ions, despite being fully expressed on the cell surface and capable of signalling upon stimulation with anti-BCR antibody as a proxy for exogenous ligand. This result is in sharp contrast with the behaviour of all other CLL-derived BCRs so far characterised in this system that could activate a cell-autonomous signal as evidenced from a persistent Ca 2+ ion influx in the transduced cells without requiring external stimuli. Hence, subset #1 BCRs differ from the so far characterized BCRs from CLL cases belonging to other stereotyped subsets, including clinically aggressive subset #2 and its satellite subset #169 as well as the clinically indolent stereotyped subset #4. In conclusion, our combined biochemical and cellular analysis demonstrates that a constitutively active cell-autonomous signalling may not be a unifying feature of all CLL clones as previously proposed, and that other mechanisms may be at the roots of the BCR-mediated signal required for leukemic cell proliferation and development of CLL.

Other malignancies in the history of CLL: an international multicenter study conducted by ERIC, the European Research Initiative on CLL, in HARMONY

Patients with chronic lymphocytic leukemia (CLL) have a higher risk of developing other malignancies (OMs) compared to the general population. However, the impact of CLL-related risk factors and CLL-directed treatment is still unclear and represents the focus of this work. We conducted a retrospective international multicenter study to assess the incidence of OMs and detect potential risk factors in 19,705 patients with CLL, small lymphocytic lymphoma, or high-count CLL-like monoclonal B-cell lymphocytosis, diagnosed between 2000 and 2016. Data collection took place between October 2020 and March2022. In 129,254 years of follow-up after CLL diagnosis, 3513 OMs were diagnosed (27.2 OMs/1000 person-years). The most common hematological OMs were Richter transformation, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Non-melanoma skin (NMSC) and prostate cancers were the most common solid tumors (STs).The only predictor for MDS and AML development was treatment with fludarabine and cyclophosphamide with/without rituximab (FC±R) (OR=3.7; 95% CI=2.79-4.91; p<0.001). STs were more frequent in males and patients with unmutated immunoglobulin heavy variable genes (OR=1.77; 95% CI=1.49-2.11; p<0.001/OR=1.89; 95% CI=1.6-2.24; p<0.001).CLL-directed treatment was associated with non-melanoma skin and prostate cancers (OR=1.8; 95% CI=1.36-2.41; p<0.001/OR=2.11; 95% CI=1.12-3.97; p=0.021). In contrast, breast cancers were more frequent in untreated patients (OR=0.17; 95% CI=0.08-0.33; p<0.001).Patients with CLL and an OM had inferior overall survival (OS) than those without. AML and MDS conferred the worst OS (p<0.001). OMs in CLL impact on OS. Treatment for CLL increased the risk for AML/MDS, prostate cancer, and NMSC. FCR was associated with increased risk for AML/MDS. AbbVie, and EU/EFPIAInnovative Medicines Initiative Joint Undertaking HARMONY grant n° 116026.

Molecular characteristics in Chinese with chronic lymphocytic leukemia by next-generation sequencing: A single-center retrospective analysis.

Although the prevalence of Asian chronic lymphocytic leukemia (CLL) patients is not as high as that of Caucasians, there are more atypical CLLs in Asia whose genetic characteristics and their clinical significance are distinct and remain unclear. A retrospective analysis of 85 CLL samples in our center was conducted from 2019 to 2022. We used next-generation sequencing with a 172 gene panel to explore the multi-gene mutational data and the mutational status of immunoglobulin heavy variable (IGHV) gene. MYD88 (20.0%) was the most frequently mutated gene, much higher than in Europe, followed in order by TP53 (18.8%), NOTCH1 (14.1%), IGLL5 (11.8%), and DNMT3A (8.2%). In addition, the incidence of ATM and SF3B1 mutations was relatively lower in our centre compared to Europe. Mutated (M)-IGHV patients were more likely to have a cooccurrence of MYD88 mutation, while complex karyotype and DNMT3A mutation were more common in the unmutated (U)-IGHV group. MYD88 mutated CLL was characterized by prevalence in young males in high-risk staging, with isolated 13q deletion and concomitant mutation of IGLL5. CLL patients with MYD88 and TP53 mutation showed an unfavorable prognosis. These results would be valuable in helping to understand the characteristics and significance of cytogenetic genetics in Chinese patients with CLL.

Subgroup-specific gene expression profiles and mixed epistasis in chronic lymphocytic leukemia.

Understanding the molecular and phenotypic heterogeneity of cancer is a prerequisite for effective treatment. For chronic lymphocytic leukemia (CLL), recurrent genetic driver events have been extensively cataloged, but this does not suffice to explain the disease's diverse course. Here, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major, orthogonal axes of gene expression variation: the first one represented the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, the three-group stratification of CLL by global DNA methylation. The second axis aligned with trisomy 12 status and affected chemokine, MAPK and mTOR signaling. We discovered nonadditive effects (epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion, suggesting that molecular understanding of disease heterogeneity requires studying such genetic events not only individually but in combination. We detected strong differentially expressed gene signatures associated with major gene mutations and copy-number aberrations including SF3B1, BRAF and TP53, as well as del(17)(p13), del(13)(q14) and del(11)(q22.3) beyond dosage effect. Our study reveals previously underappreciated gene expression signatures for the major molecular subtypes in CLL and the presence of epistasis between them.

A novel next-generation sequencing capture-based strategy to report somatic hypermutation status using genomic regions downstream to immunoglobulin rearrangements.

The somatic hypermutation (SHM) status of the clonotypic, rearranged immunoglobulin heavy variable (IGHV) gene is an established prognostic and predictive marker in chronic lymphocytic leukemia (CLL). Analysis of SHM is generally performed by polymerase chain reaction (PCR)-amplification of clonal IGHV-IGHD-IGHJ gene rearrangements followed by sequencing to identify IGHV gene sequences and germline identity. Targeted-hybridization next-generation sequencing (NGS) can simultaneously assess clonality and other genetic aberrations. However, it has limitations for SHM analysis due to sequence similarity between different IGHV genes and mutations introduced by SHM, which can affect alignment efficiency and accuracy. We developed a novel SHM assessment strategy using a targeted-hybridization NGS approach (EuroClonality- NDC assay) and applied it to 331 samples of lymphoproliferative disorder (LPD). Our strategy focuses on analyzing the sequence downstream to the clonotypic, rearranged IGHJ gene up to the IGHM enhancer (IGHJ-E) which provides more accurate alignment. Overall, 84/95 (88.4%) CLL cases with conventional SHM data showed concordant SHM status, increasing to 91.6% when excluding borderline cases. Additionally, IGHJ-E mutation analysis in a wide range of pre- and post-germinal center LPD showed significant correlation with differentiation and lineage status, suggesting that IGHJ-E analysis is a promising surrogate marker enabling SHM to be reported using NGS-capture strategies and whole genome sequencing.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3–9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Evidence of somatic hypermutation in the antigen binding sites of patients with CLL harboring IGHV genes with 100% germline identity.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

Distinct Immunogenetic Profiles of Chronic Lymphocytic Leukemia in Asia: A Taiwan Cooperative Oncology Group Registry Study

Asian patientswith chronic lymphocytic leukemia (CLL) exhibit immunoglobulin heavy variable (IGHV) gene repertoires that are distinct from those observed in Western populations, and a higher proportion of Asian CLL patients carry heavy loads of somatic hypermutations (SHM) within the B-cell receptor immunoglobulins (BcR IG). Due to the low regional incidence of CLL in Asia, only a limited number of studies had attempted to probe the phenomenon of BcR IG stereotypy in Asian populations. In this study, we analyzed the IGHV-IGHD-IGHJ gene rearrangements from a series of 255 CLL patients recruited in a nationwide, multicenter study in Taiwan. Our analysis revealed that the IGHV gene repertoire was characterized by evident biases, with IGHV3-7, IGHV4-34, and IGHV3-23 being the most frequent rearranged IGHV genes, and a higher proportion of cases carrying mutated IGHV. In terms of BcR stereotypy, the incidence of major subsets was less frequent in this cohort, with subsets #77 and #28A being the most common, while the incidence of minor subsets was approximately equivalent to that reported in the Western cohorts. With this study, we provide evidence that CLL in Asia is indeed associated with distinct immunogenetic characteristics regarding IGHV gene usage, SHM status, and BcR IG stereotypy.

Nanopore sequencing approach for immunoglobulin gene analysis in chronic lymphocytic leukemia

The evaluation of the somatic hypermutation of the clonotypic immunoglobulin heavy variable gene has become essential in the therapeutic management in chronic lymphocytic leukemia patients. European Research Initiative on Chronic Lymphocytic Leukemia promotes good practices and standardized approaches to this assay but often they are labor-intensive, technically complex, with limited in scalability. The use of next-generation sequencing in this analysis has been widely tested, showing comparable accuracy and distinct advantages. However, the adoption of the next generation sequencing requires a high sample number (run batching) to be economically convenient, which could lead to a longer turnaround time. Here we present data from nanopore sequencing for the somatic hypermutation evaluation compared to the standard method. Our results show that nanopore sequencing is suitable for immunoglobulin heavy variable gene mutational analysis in terms of sensitivity, accuracy, simplicity of analysis and is less time-consuming. Moreover, our work showed that the development of an appropriate data analysis pipeline could lower the nanopore sequencing error rate attitude.

The Biology of Chronic Lymphocytic Leukemia: Diagnostic and Prognostic Implications.

The high degree of clinical heterogeneity of chronic lymphocytic leukemia (CLL) is influenced by the disease molecular complexity. Genetic studies have allowed to better understand CLL biology and to identify molecular biomarkers of clinical relevance. TP53 disruption represents the strongest prognosticator of chemorefractoriness and indicates the use of Bruton tyrosine kinase inhibitors (BTKis) and BCL2 inhibitors. Unmutated IGHV (immunoglobulin heavy variable) genes also predict refractoriness to chemoimmunotherapy; importantly, when treated with B-cell receptor inhibitors or BCL2 inhibitors, IGHV unmutated patients display an outcome similar to that of IGHV mutated CLL. Before choosing treatment, a comprehensive assessment of TP53 and IGHV status is recommended by all guidelines for CLL clinical management. In case of fixed-duration therapeutic strategies, monitoring of minimal residual disease may provide a tool to decide treatment duration. The current precision medicine management of CLL patients might be further improved by the adoption of novel biomarkers that are emerging as clinically meaningful for this disease.

Different Time-Dependent Risk for Disease Evolution in Chronic Lymphocytic Leukemia with Distinct IGHV Gene Somatic Hypermutation Status

A great challenge for early stage patients with chronic lymphocytic leukemia (CLL) is to determine if and, consequently, when they will require treatment, highlighting the need for robust prognostic assessment. Today, immunogenetic and genomic features are considered essential for accurate risk stratification in CLL. However, when utilizing such biomarkers, the prognosis is assessed assuming stable predictability over the disease course. Here, we assessed the risk for CLL evolution overtime by evaluating time-to-first-treatment (TTFT) for 2366 CLL patients not requiring treatment at the time of diagnosis (Binet A: n=1900, 80.3%/Binet B: n=287, 12.1%), hypothesizing that risk evolution might depend on both time and the biological characteristics of the clone. Based on the somatic hypermutation status (SHM) of the immunoglobulin heavy variable genes, 1364 (58%) and 1002 (42%) patients were classified as mutated (M-CLL) and unmutated CLL (U-CLL), respectively. Regarding genomic subgroups of CLL, we focused on high risk patients carrying aberrations within the TP53 gene (TP53 abn, deletion of chromosome 17p and/or TP53 mutations) and low risk patients exhibiting isolated deletion of chromosome 13q or normal FISH according to the Döhner hierarchical model (del(13q)/normal FISH).First, we estimated the risk overtime by the hazard curve. This enables to better visualize changes in risk overtime compared to a Kaplan-Meier curve by showing the estimated proportion of cases receiving treatment in a defined interval, given that a person is still treatment-free at the start of this interval. An interpolation method was then used to estimate the values of the hazard curve at each distinct year from the time of diagnosis, and the corresponding differences in hazard values between M-CLL and U-CLL. These differences constituted the basis for the overtime analysis. In particular, the overall time frame was divided in 5-year intervals, and the distributions of differences in hazard were statistically compared between consecutive 5-year intervals. P-values less than 0.05 would indicate different evolution for the hazard curve overtime for the M-CLL and U-CLL.First, we assessed all Binet A patients, regardless of their genomic aberrations background, and found significant differences between M-CLL vs U-CLL with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, p(15,20]Vs(20,25]=0.014, and p(overall)<0.001, reflecting the constant decrease of the distance between the M-CLL and U-CLL hazard curves (Fig 1a), deemed as statistically significant between consecutive 5-year intervals. Next, we focused on different genomic risk subgroups and found that del(13q)/normal FISH patients followed the M-CLL/U-CLL rule with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, and p(overall)<0.001, indicating little additional impact on risk evolution beyond that dictated by the SHM status (Fig 1b). In sharp contrast, within TP53 abn patients we found p[0,5]Vs(5,10]=0.006, which reflects the statistically significant increase of the distance between the two hazard curves for M-CLL and U-CLL after the 5th year (Fig 1c), hence indicating decreasing risk for TP53 abn M-CLL and, vice versa, constantly increasing risk for TP53 abn U-CLL after the 5th year. For Binet B patients, we did not observe any significant differences with p[0,5]Vs(5,10]=0.2, highlighting a similar hazard evolution for M-CLL and U-CLL (Fig 1d). Low numbers precluded assessing the impact of genomic aberrations within this subgroup of patients.In conclusion, within all early stage patients as well as in subgroup analysis of high or low genomic risk Binet A patients, M-CLL was found to display a rather stable overtime risk for TTFT, independently of the presence of TP53 abn. This sharply contrasted U-CLL patients, where the overtime risk for TTFT was clearly influenced by the genomic background since del(13q)/normal FISH patients showed a similar pattern with M-CLL whereas TP53 abn patients exhibited a continuously increasing risk for disease progression. From a different standpoint, these findings corroborate the concept that the precise prognostic value of TP53 abn depends on the biological background of the CLL clone that is altogether different in patients with distinct SHM status. [Display omitted] DisclosuresStrefford:Roche: Research Funding. Delgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau. Stavroyianni:Janssen Pharmaceuticals: Honoraria; Gilead Sciences: Honoraria; Takeda: Honoraria. Gaidano:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Novartis SA: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Association of an IGHV3-66 gene variant with Kawasaki disease.

In a meta-analysis of three GWAS for susceptibility to Kawasaki disease (KD) conducted in Japan, Korea, and Taiwan and follow-up studies with a total of 11,265 subjects (3428 cases and 7837 controls), a significantly associated SNV in the immunoglobulin heavy variable gene (IGHV) cluster in 14q33.32 was identified (rs4774175; OR = 1.20, P = 6.0 × 10−9). Investigation of nonsynonymous SNVs of the IGHV cluster in 9335 Japanese subjects identified the C allele of rs6423677, located in IGHV3-66, as the most significant reproducible association (OR = 1.25, P = 6.8 × 10−10 in 3603 cases and 5731 controls). We observed highly skewed allelic usage of IGHV3-66, wherein the rs6423677 A allele was nearly abolished in the transcripts in peripheral blood mononuclear cells of both KD patients and healthy adults. Association of the high-expression allele with KD strongly indicates some active roles of B-cells or endogenous immunoglobulins in the disease pathogenesis. Considering that significant association of SNVs in the IGHV region with disease susceptibility was previously known only for rheumatic heart disease (RHD), a complication of acute rheumatic fever (ARF), these observations suggest that common B-cell related mechanisms may mediate the symptomology of KD and ARF as well as RHD.

International prognostic score for asymptomatic early-stage chronic lymphocytic leukemia

AbstractMost patients with chronic lymphocytic leukemia (CLL) are diagnosed with early-stage disease and managed with active surveillance. The individual course of patients with early-stage CLL is heterogeneous, and their probability of needing treatment is hardly anticipated at diagnosis. We aimed at developing an international prognostic score to predict time to first treatment (TTFT) in patients with CLL with early, asymptomatic disease (International Prognostic Score for Early-stage CLL [IPS-E]). Individual patient data from 11 international cohorts of patients with early-stage CLL (n = 4933) were analyzed to build and validate the prognostic score. Three covariates were consistently and independently correlated with TTFT: unmutated immunoglobulin heavy variable gene (IGHV), absolute lymphocyte count higher than 15 × 109/L, and presence of palpable lymph nodes. The IPS-E was the sum of the covariates (1 point each), and separated low-risk (score 0), intermediate-risk (score 1), and high-risk (score 2-3) patients showing a distinct TTFT. The score accuracy was validated in 9 cohorts staged by the Binet system and 1 cohort staged by the Rai system. The C-index was 0.74 in the training series and 0.70 in the aggregate of validation series. By meta-analysis of the training and validation cohorts, the 5-year cumulative risk for treatment start was 8.4%, 28.4%, and 61.2% among low-risk, intermediate-risk, and high-risk patients, respectively. The IPS-E is a simple and robust prognostic model that predicts the likelihood of treatment requirement in patients with early-stage CLL. The IPS-E can be useful in clinical management and in the design of early intervention clinical trials.

Primary vitreoretinal lymphomas display a remarkably restricted immunoglobulin gene repertoire

Primary vitreoretinal lymphoma (PVRL) is a high-grade lymphoma affecting the vitreous and/or the retina. The vast majority of cases are histopathologically classified as diffuse large B-cell lymphoma (DLBCL) and considered a subtype of primary central nervous system lymphoma (PCNSL). To obtain more insight into the ontogenetic relationship between PVRL and PCNSL, we adopted an immunogenetic perspective and explored the respective immunoglobulin gene repertoire profiles from 55 PVRL cases and 48 PCNSL cases. In addition, considering that both entities are predominantly related to activated B-cell (ABC) DLBCL, we compared their repertoire with that of publicly available 262 immunoglobulin heavy variable domain gene rearrangement sequences from systemic ABC-type DLBCLs. PVRL displayed a strikingly biased repertoire, with the IGHV4-34 gene being used in 63.6% of cases, which was significantly higher than in PCNSL (34.7%) or in DLBCL (30.2%). Further repertoire bias was evident by (1) restricted associations of IGHV4-34 expressing heavy chains, with κ light chains utilizing the IGKV3-20/IGKJ1 gene pair, including 5 cases with quasi-identical sequences, and (2) the presence of a subset of stereotyped IGHV3-7 rearrangements. All PVRL IGHV sequences were highly mutated, with evidence of antigen selection and ongoing mutations. Finally, half of PVRL and PCNSL cases carried the MYD88 L265P mutation, which was present in all 4 PVRL cases with stereotyped IGHV3-7 rearrangements. In conclusion, the massive bias in the immunoglobulin gene repertoire of PVRL delineates it from PCNSL and points to antigen selection as a major driving force in their development.

Precision Medicine Management of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in western countries, with an incidence of approximately 5.1/100,000 new cases per year. Some patients may never require treatment, whereas others relapse early after front line therapeutic approaches. Recent whole genome and whole exome sequencing studies have allowed a better understanding of CLL pathogenesis and the identification of genetic lesions with potential clinical relevance. Consistently, precision medicine plays a pivotal role in the treatment algorithm of CLL, since the integration of molecular biomarkers with the clinical features of the disease may guide treatment choices. Most CLL patients present at the time of diagnosis with an early stage disease and are managed with a watch and wait strategy. For CLL patients requiring therapy, the CLL treatment armamentarium includes both chemoimmunotherapy strategies and biological drugs. The efficacy of these treatment strategies relies upon specific molecular features of the disease. TP53 disruption (including both TP53 mutation and 17p deletion) is the strongest predictor of chemo-refractoriness, and the assessment of TP53 status is the first and most important decisional node in the first line treatment algorithm. The presence of TP53 disruption mandates treatment with biological drugs that inhibit the B cell receptor or, alternatively, the B-cell lymphoma 2 (BCL2) pathway and can, at least in part, circumvent the chemorefractoriness of TP53-disrupted patients. Beside TP53 disruption, the mutational status of immunoglobulin heavy variable (IGHV) genes also helps clinicians to improve treatment tailoring. In fact, patients carrying mutated IGHV genes in the absence of TP53 disruption experience a long-lasting and durable response to chemoimmunotherapy after fludarabine, cyclophosphamide, and rituximab (FCR) treatment with a survival superimposable to that of a matched general population. In contrast, patients with unmutated IGHV genes respond poorly to chemoimmunotherapy and deserve treatment with B cell receptor inhibitors. Minimal residual disease is also emerging as a relevant biomarker with potential clinical implications. Overall, precision medicine is now a mainstay in the management and treatment stratification of CLL. The identification of novel predictive biomarkers will allow further improvements in the treatment tailoring of this leukemia.

Klinički značaj aberacija TP53 gena i IGHV mutacionog statusa u hroničnoj limfocitnoj leukemiji

Chronic lymphocytic leukemia (CLL) is a very heterogeneous disease with a variable clinical course. On the one side of the spectrum, there are patients with aggressive and resistant disease, of which they die only a few months after diagnosis while, on the other side, there are patients with an indolent, slowly progressive disease that does not require treatment for decades. The reasons for this are only partially known, but they have been the subject of numerous scientific studies during the last several decades. Consequently, the concept of prognostic and predictive factors in CLL have emerged, which aims to predict the clinical course and its therapeutic outcome. With the improvement of understanding the pathophysiology of this disease, the lists of prognostic and predictive factors are getting longer every year, but they also overlap. In this review, we singled out the aberrations of the TP53 gene and the IGHV (immunoglobulin heavy variable) gene mutational status as the two most important and most studied factors that have both prognostic and predictive significance.

Prognostic impact of prevalent chronic lymphocytic leukemia stereotyped subsets: analysis within prospective clinical trials of the German CLL Study Group (GCLLSG).

Almost one-third of all patients with chronic lymphocytic leukemia (CLL) express stereotyped B-cell receptor immunoglobulins (BcR IG) and can be assigned to distinct subsets, each with a particular BcR IG. The largest stereotyped subsets are #1, #2, #4 and #8, associated with specific clinico-biological characteristics and outcomes in retrospective studies. We assessed the associations and prognostic value of these BcR IG in prospective multicenter clinical trials reflective of two different clinical situations: (i) early-stage patients ('watch and wait' arm of the CLL1 trial) (n=592); (ii) patients in need of treatment, enrolled in three phase III trials (CLL8, CLL10, CLL11), treated with different chemo-immunotherapies (n=1,861). Subset #1 was associated with del(11q), higher CLL International Prognostic Index (CLL-IPI) scores and similar clinical course to CLL with unmutated immunoglobulin heavy variable (IGHV) genes (U-CLL) in both early and advanced stage groups. IGHV-mutated (M-CLL) subset #2 cases had shorter time-to-first-treatment (TTFT) versus other M-CLL cases in the early-stage cohort (hazard ratio [HR]: 4.2, confidence interval [CI]: 2-8.6, P<0.001), and shorter time-to-next-treatment (TTNT) in the advancedstage cohort (HR: 2, CI: 1.2-3.3, P=0.005). M-CLL subset #4 was associated with lower CLL-IPI scores and younger age at diagnosis; in both cohorts, these patients showed a trend towards better outcomes versus other MCLL. U-CLL subset #8 was associated with trisomy 12. Overall, this study shows that major stereotyped subsets have distinctive characteristics. For the first time in prospective multicenter clinical trials, subset #2 appeared as an independent prognostic factor for earlier TTFT and TTNT and should be proposed for risk stratification of patients. (Trials registered at clinical trials. gov identifiers: NCT00262782, NCT00281918, NCT2000769522, and NCT01010061).