Search

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

Together with multicolor flow cytometry, quantitation of clonal immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements represents the current standard for the detection of minimal / measurable residual disease (MRD) in treatment protocols for pediatric acute lymphoblastic leukemia (ALL) patients. Despite the adoption of next generation sequencing (NGS) in the routine identification of clonal IG/TR gene rearrangements as markers for MRD detection, real-time quantitative (q)PCR is still the standard for MRD quantitation in follow-up samples. So far, no large-scale direct comparison of qPCR- and NGS-based MRD quantitation has been performed. We compared qPCR- and NGS-MRD evaluation in a cohort of children with B-cell precursor (BCP) ALL treated on the AIEOP-BFM ALL 2009 protocol and assessed the feasibility and relevance of this method for the stratification at day 33 (EOI). In total, 459 patients were diagnosed with BCP-ALL from 2010 to 2018, and 437 of them were included in our study based on the availability of residual DNA material isolated from day 33 bone marrow aspirates and having at least one IG/TR MRD marker detectable by standard qPCR with protocol-required sensitivity of 10 -4. Sequencing libraries were prepared according to the EuroClonality-NGS group SOP (Brüggemann et al, Leukemia 2019) with the total DNA input normalized to the equivalent of 150,000 nucleated cells to reach MRD sensitivity of 10 -5 and sequenced on Illumina NovaSeq and MiSeq instruments. In total of 780 IG/TR markers evaluated by both NGS and qPCR. Sequencing data were analyzed using the ARResT/Interrogate (Bystry et at, Bioinformatics 2017) pipeline and a custom bioinformatic analysis process and the NGS-MRD results were normalized to the EuroClonality-NGS central in-tube quality/quantification control (cIT-QC; Knecht et al, Leukemia 2019). From the total 780 IG/TR MRD markers evaluated by both methods, 629 (80.6%) were concordant with 242 markers being MRD positive and 387 negative. From 82 markers that were only positive by qPCR and not by NGS, 76 were positive below the quantitative range (positive non-quantifiable). Specificity analysis was performed for each marker by searching for the junction sequence across the dataset of all patients' NGS results. Based on these results, 22 out of 82 markers positive only by qPCR were classified as potentially unspecific (false positive) and similarly 32 unspecific markers were identified among the 69 positive only by NGS. This was also supported by unspecific amplification of the polyclonal control in 27 out of these 32 corresponding qPCR systems, in some cases leading to qPCR negative classification determined by the EuroMRD guidelines. Overall stratification of patients based only on day 33 MRD by qPCR or NGS was concordant in 76% of patients by both methods, while in 19% of patients, NGS-MRD quantitation led to the assignment to a lower-risk group, mainly due to the elimination of false-positive results. Furthermore, analysis of all positive markers across all patients' NGS libraries showed, that one out of 10 markers (mainly in the IGK, TRG and TRD loci) used for qPCR-MRD stratification did not provide satisfactory specificity, although they fully met EuroMRD criteria during the optimization of qPCR patient-specific assays. Our results show that NGS-MRD is highly concordant with traditional qPCR-based strategy and has comparable sensitivity and clinical value in the setting of a BFM-based clinical protocol, while being less laborious and providing significantly more specific results and additional information on the IG/TR repertoire (Kotrova et al, Blood 2016). Our study also emphasizes the importance of selecting MRD markers of adequate specificity at diagnosis. Currently, this selection can be assisted by these broad sequencing data on IG/TR repertoire of large number of patients. Based on these results, we propose that frontline NGS-MRD evaluation developed by the EuroClonality-NGS working group can be used as an alternative to traditional qPCR-based MRD quantitation in future MRD-based treatment protocols. Supported by grants NU20-03-00284 and NU20-07-00322 from the Czech Health Research Council and 534120 from Charles University. All methods were established through collaboration within the EuroClonality-NGS and EuroMRD groups. Disclosures van der Velden: Agilent: Research Funding; Navigate: Other: Service Level Agreement; Janssen: Other: Service Level Agreement; EuroFlow: Other: Service Level Agreement, Patents & Royalties: for network, not personally; BD Biosciences: Other: Service Level Agreement. Brüggemann: Incyte: Other: Advisory Board; Janssen: Speakers Bureau; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau. Langerak: Erasmus MS, University Medical Center: Current Employment; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Gilead: Research Funding; Janssen: Speakers Bureau.

Introduction: At a median follow-up of 5 years (cutoff of May 8, 2020), the MURANO trial (NCT02005471) demonstrated deeper responses, including superior rates of undetectable (u)MRD and progression-free survival, in patients (pts) with relapsed/refractory (R/R) CLL treated with fixed-duration VenR vs bendamustine-rituximab (BR). With longitudinal MRD assessments in peripheral blood (PB), we now report kinetics of MRD growth after end of treatment (EOT), and the clinical and molecular factors associated with MRD growth rates. Methods: Pts were randomized to VenR (venetoclax 400mg daily for 2 years + standard-dose rituximab for the first 6 months) or BR (6 months). PB MRD samples were collected every 3-6 months and analyzed centrally by allele-specific oligonucleotide-polymerase chain reaction and/or flow cytometry; uMRD threshold was For longitudinal analysis of MRD growth dynamics, a population-based logistic growth model with a nonlinear mixed effects approach was developed as previously described (Al-Sawaf et al. EHA 2021). Pts who completed treatment (Tx) without progressive disease (PD) and who had ≥2 measurable time points post-EOT were included; data below the lower limit of quantification in those pts was handled by a likelihood-based method (Bergstrand & Karlsson, AAPS J 2009). Prognostic markers and patient demographics were screened as covariates for impact on key model parameters, based on statistical and graphical assessments. Variables with >20% missing data or with low representation ( Results: Overall, 284 pts completed the study Tx without PD. Pts with no MRD data (n=23), pts with MRD data only on-Tx (n=6) or only after PD/next-line of Tx (n=2), or pts with The median MRD level at EOT was significantly lower after VenR (1.88 x 10 −5) than BR (7.06 x 10 −4; p=5.1 x 10 −8, Figure 1A). In the VenR arm, no statistically significant difference in median MRD level at EOT was seen in pts with mutated or unmutated immunoglobulin heavy chain variable gene (IGHV), or pts with or without TP53 mutation (Table 1). However, in the BR arm, pts with TP53 mutation had significantly higher median MRD levels at EOT than those with TP53 wild-type (Table 1). Overall, median MRD doubling time post-EOT was significantly longer for pts treated with VenR (93 days; n=91) than BR (53 days; n=120; p=1.2 x 10 −7, Figure 1B). Based on covariate screening of 27 prognostic markers and patient demographics, age (< or ≥65 years), VenR Tx, IGHV status, TP53 mutation status, and disease burden at study initiation (low/medium risk of tumor lysis syndrome vs high risk) showed significant impact on MRD growth rate (Table 1). The median MRD doubling time was 53 days in pts aged Conclusion: Using a robust, population-based model of MRD growth dynamics, we demonstrate that post-EOT MRD growth was slower in pts with R/R CLL treated with VenR compared with BR. Together with similar observations in the frontline setting of venetoclax-obinutuzumab Tx, these results suggest that, compared with chemoimmunotherapy, fixed-duration venetoclax plus anti-CD20 therapy may contribute to prolonged CLL disease control by slowing the growth of the residual disease clones. It also implies that clonal growth may be influenced by biological and Tx-related parameters. The underlying mechanisms need to be further explored. Figure 1 Figure 1. Disclosures Kater: Abbvie: Honoraria, Other: Ad Board, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee. Lu: Genentech, Inc.: Current Employment; F. Hoffmann-La Roche Ltd: Current holder of individual stocks in a privately-held company. Langerak: Erasmus MS, University Medical Center: Current Employment; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Gilead: Research Funding; Janssen: Speakers Bureau. Mellink: Cytogenetic Field: Consultancy; Genome Diagnostics Laboratory, AUMC: Current Employment; Financial support related to microarray analysis of Murano samples: Research Funding. Chyla: AbbVie: Current Employment, Current equity holder in publicly-traded company. Wu: Genentech, Inc.: Current Employment; Roche/GNE: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Boyer: Roche: Current Employment. Lefebure: F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company; Roche: Current Employment. Jiang: F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company; Genentech, Inc./F.Hoffmann-La Roche Ltd: Current Employment. Seymour: Sunesis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mei Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction: In the MURANO trial (NCT02005471), fixed-duration VenR (2 yrs Ven + R for the first 6 mo) improved survival and rates of undetectable minimal residual disease (MRD) over bendamustine (Benda)-R in pts with R/R CLL. Novel recurrent mutations in BCL2 and family genes (eg BAX and PMAIP1 [encoding Noxa]) have been linked to Ven resistance. We analyzed MRD+ samples from VenR-treated pts in MURANO to assess the nature and frequency of acquired mutations in BCL2 family genes (marker of emergent Ven resistance) and TP53 (may negatively impact response to re-Tx). We also analyzed the impact of these mutations on time to next CLL Tx (TTNT) and subsequent response. Methods: Pts included in this analysis received up to 2 yrs of VenR and had, at any time, ≥1 CLL cell per 100 leukocytes (high MRD+) in peripheral blood. Deep next generation sequencing (NGS) was performed without cell selection using a custom targeted NGS panel, encompassing 87 cell death related genes (coding regions and selected promoters). A CD19-enriched pre-Tx sample, analyzed by whole-exome sequencing, was available for all pts for comparison. Data cutoff was May 8, 2020, for the MURANO main study and Oct 13, 2020, for the substudy (where pts received next-line VenR following progression after initial VenR Tx). TTNT was calculated using the main study Ven completion/discontinuation date and the date of the first subsequent CLL Tx. Results: 107 samples from 42 pts, collected 21-42 mo post-Tx initiation, were evaluable. No BCL2 mutations were identified (limit of detection - variant allele frequency [VAF] 1%). Acquired BAX and PMAIP1 mutations were seen in 4 and 2 pts, respectively. Discordance between VAF and CLL MRD, and lack of proportionality in results with serial testing, suggested presence of BAX mutations in the non-CLL compartment in some pts, with further testing of sorted populations needed to clarify the source. All BAX mutations identified have been predicted to lead to loss of protein function, including 4 frameshift mutations. PMAIP1 mutations included a frameshift mutation (T86fs) and a mutation affecting gene splicing, both suggesting a loss-of-function effect. Nineteen TP53 mutations in 15/42 pts were observed across longitudinal samples (8/19 mutations in 6 pts present at baseline, 11/19 in 11 pts newly acquired, and 2 pts with both baseline and acquired mutations). Co-occurrence of TP53mut with BAXmut or PMAIP1mut was observed in 3/15 and 1/15 pts, respectively; based on VAF and assuming heterozygosity, these were likely in different clones. After Ven cessation, 28/42 (66.7%) had received an additional anti-CLL Tx by the cutoff; 19 pts received Ven-based regimens (15 in the substudy; 4 received other Ven-containing regimens per investigators' choice), 8 pts received ibrutinib (Ibr) monotherapy and 1 pt received Benda. Of the 19 pts who received Ven-based regimens (median 22.6 mo [range 11.9-38.1] post-main study end of Tx), 8 had TP53mut (5 acquired, 3 baseline), with 5/8 (63%) responding (complete response, 3 pts; partial response [PR], 2 pts). Six/11 pts (55%) with TP53-wild-type (WT) responded, all PRs. Two pts with TP53mut received Ibr as next Tx (one responded) vs 6 TP53-WT pts (all responded). The one pt treated with Benda without response had coexisting TP53mut and BAXmut. One patient with BAXmut received Ven re-Tx without response, while in pts with BAXmut and PMAIP1mut, responses to Ibr were 100% (2/2 pts) and 50% (1/2 pts), respectively (Table). No association was apparent between acquired TP53mut and TTNT, median 784 days (d; range 248-1051) vs 617 d (range 302-1142) in TP53-WT pts. Pts harboring BAXmut (n=5) had a shorter median TTNT (305 d; range 248-874) than BAX-WT pts (678 d; range 302-1142). Conclusion: No acquired BCL2 mutations were found in this analysis of pts from MURANO who received fixed-duration VenR Tx and subsequently manifest high MRD+. This suggests that fixed-duration Tx may reduce the propensity for selection of BCL2 resistance mutations, although confirmation using orthogonal methodology with higher sensitivity is required. Acquired TP53 mutations, noted in ~40% of pts, did not preclude response to subsequent Tx (primarily Ven-based regimens). Consistent with the TP53-independent mechanism of Ven, Ven re-Tx may be effective for pts with MRD+ disease who have relapsed following initial fixed-duration VenR Tx. More data are needed to better understand the significance of BAX or PMAIP1 mutations. Figure 1 Figure 1. Disclosures Seymour: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mei Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sunesis: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau. Wu: Roche/GNE: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Genentech, Inc.: Current Employment. Popovic: AbbVie: Current Employment, Current equity holder in publicly-traded company. Eichhorst: Consultant Department I for Internal Medicine: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Adaptive Biotechnologies: Speakers Bureau; Hexal: Speakers Bureau; ArQule: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedica (UK): Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; University Hospital of Cologne: Current Employment. Hillmen: University of Leeds: Current Employment; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Research Funding; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Honoraria; SOBI: Honoraria. Kipps: European Research Initiative on CLL (ERIC): Honoraria; DAVA Pharmaceuticals: Speakers Bureau; Bionest Partner: Other; Celgene: Consultancy, Honoraria, Other, Research Funding; Genetech: Honoraria, Other; Genentech-Roche: Consultancy; Gilead Sciences: Consultancy, Honoraria, Other, Speakers Bureau; Janssen: Consultancy, Honoraria, Other, Research Funding, Speakers Bureau; Roche: Honoraria, Other; MD Anderson Cancer Center: Research Funding; Velos: Research Funding; CRIM: Research Funding; Indy Hematology Review: Other; TG Therapeutics: Other; Verstem: Other, Speakers Bureau; University of California, San Diego: Current Employment; Pharmacyclics/AbbVie: Honoraria, Research Funding; Breast Cancer Research Foundation: Research Funding; SCOR - The Leukemia and Lymphoma Society: Research Funding; National Cancer Institute/NIH: Honoraria, Research Funding; DAVAOncology: Consultancy, Honoraria, Other; AbbVie: Consultancy, Honoraria, Other, Speakers Bureau; Oncternal Therapeutics, Inc.: Current holder of stock options in a privately-held company, Other: Stock or other ownership, Patents & Royalties: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory., Research Funding; Genentech/Roche: Honoraria; Moores Cancer Center: Current Employment; MedImmune Inc: Research Funding; GlaxoSmithKline: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics LLC, an Abbvie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbott Laboratories: Consultancy, Research Funding. Langerak: Janssen: Speakers Bureau; Gilead: Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Erasmus MS, University Medical Center: Current Employment. Owen: Incyte: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Servier: Honoraria; Pharmacyclics: Research Funding; Genentech: Research Funding; Gilead: Honoraria. Dubois: Abbvie: Research Funding; Genentech: Research Funding; Roche: Research Funding. Mellink: Cytogenetic Field: Consultancy; Genome Diagnostics Laboratory, AUMC: Current Employment; Financial support related to microarray analysis of Murano samples: Research Funding. Van Der Kevie-Kersemaekers: Amsterdam University Medical Centers: Current Employment. Dunbar: AbbVie: Current Employment. Jiang: Genentech, Inc./F.Hoffmann-La Roche Ltd: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. Chyla: AbbVie: Current Employment, Current equity holder in publicly-traded company. Boyer: Roche: Current Employment. Thadani-Mulero: Roche: Current Employment, Current equity holder in publicly-traded company. Lefebure: Roche: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. Harrup: AstraZeneca: Other: Advisory board. Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.