To the Editor:

The outcome of patients with chronic lymphocytic leukemia (CLL) is known to be influenced by the mutational status of the immunoglobulin heavy-chain variable (IGHV) region [1]. In the past decade, it has been shown that a large part of CLL share B-cell receptor (BCR) sequence homologies, defining subsets of patients with similar clinical outcome [2]. The CLL Subset#2 (S#2), defined by the presence of the stereotyped IGHV3-21-derived rearrangement, with a short third complementarity-determining region (CDR3), has been linked to unfavorable prognosis. This translates in shorter time to first treatment, independently of other prognostic factors such as IGHV mutational status or genetic alterations [3, 4]. Despite S#2 being the most prevalent subset, few studies have investigated treatment responses and progression-free survival (PFS) in this context. Chemoimmunotherapy (CIT) is unsatisfactory in this group of patients [5, 6], but responses to targeted therapies (TT) have not been investigated. The aim of this study was to evaluate the outcome of patients with S#2 compared to patients with other IGHV3-21 subsets in the era of TT.

A retrospective multicentric study was conducted across 31 French Innovative Leukemia Organization (FILO-CLL) affiliated centers in France, including patients diagnosed between 1998 and 2023. The study was declared to the Health data Hub according to French legislation. Patients identified with a productive IGHV3-21 rearrangement, available clinical and survival data, and no opposition to the study, were included. S#2, not-Subset#2 (nS#2) and IGHV mutational status were categorized according to the recommendations of the European Research Initiative on CLL (ERIC) [7]. Treatment strategies were CIT, Bruton Tyrosine Kinase inhibitors (BTKi), and B-Cell Lymphoma 2 inhibitors (BCL2i). Survival analysis, according to ERIC recommendations [7], considered borderline IGHV status as mutated. Time to first treatment (TTFT) and subsequent PFS analysis were performed using Kaplan–Meier estimation and log-rank test (R software, CRAN). Potential confounders, including age, sex, mutated (mIGHV) and unmutated (uIGHV) status, complex karyotype (CK) (≥3 abnormalities), high-CK (HCK) (≥5 abnormalities) and TP53 alterations were secondarily adjusted using propensity matching. Briefly, a propensity score-matched analysis was performed matching age, sex, IGHV status, karyotype and TP53 alteration, with a 0.2 caliper.

Of 410 patients (median age: 66 years), 225 were identified as S#2, and 185 as nS#2 (Table 1). A significantly lower proportion of uIGHV (33% vs 51%, p < 0.001) was observed in the S#2 cohort, mainly due to an increased proportion of borderline IGHV status compared to the nS#2 group (29% vs 9.7%). There we no significant difference in Binet stage, TP53 alterations, nor cytogenetic profiles between the S#2 and nS#2 groups (Table 1).

Table 1 Patient characteristics at diagnosis.
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With a median follow-up of 64 months, 176 (78%) S#2 patients required first-line therapy as follows: 113 with CIT, 45 with BTKi, 12 with BCL2i and 6 with unspecified other treatment. Among the nS#2 group, 122 (66%) patients were treated, 81 with CIT, 28 with BTKi, 11 with BCL2i and 2 with unspecified other treatment (Supplementary Table 1). Four cases lost to follow-up were excluded from survival analysis (1 in S#2 CIT, 1 in S#2 TT and 2 in nS#2 CIT group).

As expected, TTFT was shorter for the S#2 group (median TTFT: 28 months vs 40 months, p = 0.018, Supplementary Fig 1A). There was no additional impact of the IGHV status (mutated or not) within the S#2 group (p = 0.2) by contrast to the nS#2 group (p = 0.0001) (Supplementary Fig 1).

PFS differed significantly according to the type of first-line therapy in both the S#2 and nS#2 groups (p = 0.01, Fig. 1A), CIT yielding significantly poorer results. Contrasting with the S#2 group, a strong prognostic impact of IGHV status was observed in the nS#2 group both in patients receiving CIT or BTKi (Fig. 1B, D). Strikingly, S#2 patients treated with TT showed a significantly improved PFS compared to nS#2 patients (p = 0.01) (Fig. 1C) with respective estimated 5-year PFS rates of 98% vs. 77% for TT (p = 0.01). This difference persisted even when considering only patients treated with BTKi, without impact of the IGHV mutational status in the S#2 group, contrary to the nS#2 group (p = 0.041, Fig. 1D). Finally, these results translated into a statistically significant benefit for TT compared to CIT in the S#2 group but not in the nS#2 group (Fig. 1E–H).

Fig. 1: Outcomes of patients after a first line of treatment.
figure 1

Kaplan–Meier estimator of PFS; A Whole cohort according to S#2/nS#2 and treatment strategies (CIT vs TT), B patients treated by CIT according to S#2/nS#2 and IGHV status, C patients treated by TT according to S#2/nS#2 status, D patients treated by BTKi according to S#2/nS#2 and IGHV status, E nS#2 patients treated by CIT or TT, F S#2 patients treated by CIT or TT, G nS#2 patients treated by CIT, BTKi or BCL2i, H S#2 patients treated by CIT, BTKi or BCL2i.

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Remarkably, while TP53 alterations significantly altered PFS in the nS#2 cohort (p = 0.002) (Supplementary Fig. 2A), they were not associated with a prognostic impact in the S#2 cohort, whatever the type of treatment (p = 0.48, Supplementary Fig. 2B). Lastly, the high rate of TP53 alterations observed (Table 1) did not affect survival comparisons as confirmed by the results obtained after propensity matching (Supplementary Fig. 3A, B), with a persisting benefit of TT in the S#2 group compared to the nS#2 group (p = 0.044).

To minimize the potential impact of confounding factors, propensity score matching was performed on the population treated in the first line (Supplementary Table 2). After propensity matching, the PFS remained significantly different according to treatment strategies, with a persisting benefit of TT for S#2 group compared to the nS#2 group (p = 0.044, Supplementary Fig. 3B), with estimated 5-year PFS rates of 97% versus 75%, respectively. These results were independent of the IGHV status in the S#2 group, but not in the nS#2 group (Supplementary Fig. 3C, D).

Despite significant progress in CLL management over the past decade, it has remained unclear whether S#2 patients would benefit from new TT strategies. This was addressed here, in potentially the largest cohort specifically dedicated to patients with S#2 CLL, compared to nS#2 IGHV3-21 patients. Our results demonstrate that the use of TT as first-line treatment significantly improves the outcome of S#2 patients. Notably, this advantage is observed with both BTKi and BCL2i treatments compared to CIT, regardless of the IGHV mutational status. Five-year PFS in nS#2 on BTKi was 63% in uIGHV and 89% in mIGHV. This results suggest that PFS of nS#2 IGHV3-21 patients treated with BTKi is similar to previously published results and show that TT provides greater benefits to S#2 patients than other IGHV3-21 patients [8, 9].

The very favorable outcome observed with BTKi in patients with S#2 CLL can be explained by the crucial role of an autonomous BCR signaling in this subgroup of patients [10, 11]. Interestingly, it was recently shown that S#2 CLL cells harbor a BCR encoded by the IGHV3-21/IGLV3-21 gene pair, and that S#2 cell-autonomous BCR signaling is driven by a specific light chain mutation (IGLV3-21R110), conferring an aggressive behavior, regardless of its association with S#2 [12, 13]. Thus, results of this study may also suggest that patients with IGLV3-21R110 could also benefit significantly from BTKi therapy.

The impressive response to BCL2i in S#2 CLL observed here must however be interpreted with caution considering the limited number of patients treated with BCL2i as first-line therapy. Another limitation is the shorter follow-up of this population, due to a more recent regulatory approval of venetoclax in France, impairing the appreciation of relapse risk. Despite these limits, these results suggest that S#2 CLL may not only rely on the BCR signaling pathway but also exhibit a strong BCL2 dependency, which could be linked to specific biological features, as previously suggested [14]. In line with this, in the HOVON-139 trial, which evaluated the efficacy of first-line MRD-guided treatment with obinutuzumab and venetoclax in unfit CLL, patients with IGVL3-21R110 did not experience worse outcomes than other patients [15]. Nevertheless, further studies and long-term follow-up are warranted to clearly determine the better outcome of S#2 patients treated with time-limited TT, particularly those involving BCL2i.

Considering potential confounding factors, the TP53 mutation rate was higher in this study than previously reported in S#2 and nS#2 groups. However, a recent study in the IGLV3-21R110 subgroup in the HOVON-141 and HOVON-139 trials showed a comparable incidence of TP53 mutations in this population (14–27%) [15]. Here, TP53 mutation rates were well balanced between the S#2 and nS#2 groups and did not significantly impact the differences observed in terms of PFS, mainly because of the absence of any prognostic impact of TP53 status in the S#2 group.

The proportion of mIGHV did not differ significantly between the two groups. Surprisingly, there were only 38% of mIGHV in the S#2 group, while a rate of 60–65% is commonly reported. This discrepancy could be explained by the high proportion of borderline IGHV status. Hengeveld et al. recently reported similar findings with IGLV3-21R110, centered around the 98% homology cutoff. Finally, as previously published [3], the presence of a stereotyped S#2 IGHV is shown to erase the prognostic impact of the IGHV status considering either TTFT or PFS and whatever the treatment choice.

Overall, the findings reported here demonstrate that even if associated with a shorter TTFT, S#2 should no longer be regarded as a negative prognostic factor in the era of TT. Furthermore, these results support the strong theranostic impact of S#2, with exceptional responses to BTKi, leading to propose this class as the first therapeutic option, while CIT should be strictly avoided in S#2 patients. This paradigm shift underscores the importance of tailored therapeutic approaches, based on IGHV subset characteristics, alongside traditional prognostic factors, and calls for updated recommendations in the management of S#2 CLL patients.