Introduction

The last decade has seen remarkable advances in therapy of chronic lymphocytic leukemia (CLL) with the advent of combination chemotherapy (fludarabine and cyclophosphamide (FC))1, 2 and then chemoimmunotherapy with rituximab (FCR).3, 4 Indeed, a subset of low-risk patients with IGHV-mutated disease may be essentially cured by FCR, but still may suffer the toxic effects of chemotherapy. However, patients with higher-risk genetic abnormalities (IGHV unmutated) experience inferior outcomes with a median progression-free survival (PFS) of 4.2 years,5 while those with the highest-risk factor, del(17)(p13.1) [del17p], have a median PFS of ∼1 year.3 Shorter remissions particularly in high-risk subgroups, and poor survival with conventional salvage regimens after FCR relapse,6, 7 present a need for novel therapies for CLL.

Bruton’s tyrosine kinase (BTK) has emerged as an attractive target with the observation that the genetic disease, X-linked agammaglobulinemia, in which BTK is inactivated, results in the failure of B cells to develop, with few other complications outside of infectious morbidity that is clinically manageable.8 Ibrutinib is a once-daily, first-in-class, covalent inhibitor of BTK, which binds irreversibly to Cys481 in BTK with an IC50 of 0.5 nM9 allowing for once-daily, oral administration with sustained enzyme inhibition. RESONATE was a randomized comparison of ibrutinib to ofatumumab in previously treated CLL patients, many with high-risk prognostic factors. The first report of this trial demonstrated that ibrutinib significantly improved PFS, overall survival (OS), and overall response rates (ORR) compared with ofatumumab, and was acceptably tolerated,10 leading to ibrutinib’s approval for previously treated CLL and del17p CLL.

Ibrutinib shows marked efficacy in genetically high-risk CLL, particularly del17p,11 known to confer a very poor prognosis in addition to TP53 mutation.12 Whole exome sequencing studies have identified recurrent mutations particularly in NOTCH1,13 ATM, SF3B114, 15 and BIRC3.16 These mutations, enriched in previously treated CLL, are associated with poor prognosis in retrospective studies.17 In the CLL8 study, both TP53 and SF3B1 mutations were risk factors for reduced PFS, but only TP53 for reduced OS,18 and NOTCH1 mutation was associated with lack of benefit from the addition of CD20 monoclonal antibody in two studies.18, 19 In retrospective studies, NOTCH1 mutation has also been associated with increased risk of Richter transformation (RT).20, 21 Here, we report updated results with up to 2-year follow-up from the RESONATE trial, including subgroup analysis for baseline high-risk genetic features. We find that the marked benefit of ibrutinib continues and is preserved in all evaluable genetic subgroups.

Materials and methods

Study design

RESONATE is a multicenter, international, open-label, randomized, phase 3 study that compares the efficacy and safety of ibrutinib to ofatumumab, as previously described (NCT01578707).10 At the time of this update, 123 patients randomized to ofatumumab received ibrutinib in crossover. See Supplementary Appendix for details.

Patients

All patients provided written informed consent. The study was approved by the institutional review board or ethics committee at each participating institution and conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice.

Procedures

Updated efficacy data from this analysis, including PFS and ORR, were by investigator assessment. Procedural details have been described previously10 and can be found in Supplementary Appendix.

Mutation Identification

In order to make our results applicable to general practice, all mutation analyses were performed by a clinically available, FDA-approved targeted next generation sequencing panel that is being increasingly used in clinical practice, the Foundation One Heme Panel. This panel has a median sequencing depth 500X and reports variant allele frequencies of approximately 1% or higher22 (see Supplementary Appendix for details). As is typical in clinical practice, matched germline testing was not performed; known common polymorphisms were removed based on those identified in the 1000 Genomes Project (dbSNP135). Putative mutations were evaluated based on COSMIC v62; all candidate mutations of any detected allele frequency, classified as known, likely, or unknown, were counted as a mutation. Of note, 138 of 140 TP53 mutations identified were classified as known or likely to have a functional effect.

Statistical analysis

Please see Supplementary Appendix for additional details on outcomes measures.10

Results

Baseline patient characteristics

Patients in the ibrutinib (n=195) and ofatumumab (n=196) arms had a median of three and two prior therapies, respectively, with 53 vs 46% of patients receiving study therapy in the fourth line of therapy or beyond; 18% of ibrutinib- and 27% of ofatumumab-treated patients had received only one prior therapy (Table 1); approximately 32% of ibrutinib patients had del17p, and 32% had del(11)(q22.3) [del11q].

Table 1 Baseline characteristics
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Consistent with this relapsed higher-risk population, the frequencies of TP53, NOTCH1, SF3B1 and BIRC3 mutations were high compared with other studies.16, 18, 23 Most notably, 51% of ibrutinib and 46% of ofatumumab patients carried mutations in TP53. Of 195 ibrutinib patients, 154 had TP53 samples available. Of 45 ibrutinib patients with del17p and a sample available for analysis, 84% (n=38) had coexistent TP53 mutation, consistent with prior reports;24 of 79 ibrutinib patients with mutated TP53, 52% (n=41) carried this mutation in the absence of del17p. In the ibrutinib arm, 39 of 153 (25%) evaluable patients had complex karyotype (CK); of 47 del17p patients in the ibrutinib arm with karyotype data reported, 20 (43%) had CK.

Patient disposition

With a median 19-month follow-up (maximum 26 months on study), 145 (74%) patients on the ibrutinib arm continue ibrutinib on study. Fifty (26%) patients discontinued ibrutinib (19 (10%) due to progressive disease (PD), 13 (7%) due to adverse events (AE) and 10 (5%) due to death; Supplementary Table 1).

Efficacy

Ibrutinib showed significant improvement in PFS compared with ofatumumab (median not reached (NR) vs 8.1 months; HR 0.106; Figure 1a) with the benefit of ibrutinib apparent for all clinical and genetic subgroups (Supplementary Figure 1). Median PFS was NR on the ibrutinib arm in any prognostic subgroup including del17p (Figure 1b) and del11q. Second-line ibrutinib PFS outcomes were significantly improved compared with those in later lines of therapy (P=0.0348) (Figure 1c). Supplementary Figure 2 shows PFS for ibrutinib by one, two, three and ⩾four prior therapies.

Figure 1
figure 1

PFS in patient subgroups. (a) Overall PFS; P<0.0001 for the comparison of ibrutinib vs ofatumumab. (b) PFS in patients with del17p CLL; P=0.2575 for ibrutinib-treated patients with and without del17p and P=0.0582 for ofatumumab-treated patients with and without del17p. (c) PFS in patients with 1 vs >1 prior therapy; P=0.0348 for ibrutinib-treated patients with 1 prior line vs >1 prior line of therapy and P=0.2761 for ofatumumab-treated patients with 1 prior line vs >1 prior line of therapy. (d) PFS in patients who did or did not develop lymphocytosis; P=0.0259 for ibrutinib-treated patients with and without lymphocytosis at baseline and P=0.0095 for ofatumumab-treated patients with and without lymphocytosis.

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The 24-month PFS rate was 74% for the ibrutinib arm. The 18-month PFS with ibrutinib was similar regardless of baseline genetic factors including unmutated IGHV, del17p, del11q, CK or mutations including NOTCH1, BIRC3 and ATM (Table 2). With SF3B1 mutation, a trend toward a lower 18-month PFS rate was seen in the ibrutinib arm (P=0.1424); however, when all data were taken into account by log-rank test (P=0.3349), this trend was less apparent. The presence of del17p or del11q did not confer inferior PFS outcomes compared with PFS in patients with neither deletion (P=0.2160; Figure 2a). Those with any del17p/TP53 alteration (carrying either one or both abnormalities) did not show significantly inferior PFS compared with those with neither abnormality (P=0.1306; Figure 3a). Interestingly, however, an exploratory subset analysis focusing on patients with both del17p and TP53 mutation (n=38) compared with patients with neither of these abnormalities (n=68) showed worse PFS in the subset carrying both abnormalities (P=0.0381; Figure 2b). Although survival assessment by CK status was somewhat limited due to missing CK data in 22% of patients, available data suggest that PFS was not significantly different in ibrutinib patients with known CK relative to those without CK (Figure 3b). Among ofatumumab-treated patients, unmutated IGHV (P=0.0436) and presence of del11q (P=0.0654) had a lower 18-month PFS rate (Table 2) and NOTCH1 mutation was associated with a worse overall PFS (log-rank P=0.0064); conversely, the presence of a NOTCH1 mutation did not negatively impact the efficacy of ibrutinib (Table 2; Supplementary Table 2).

Table 2 18-month PFS rate and ORR by subgroup
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Figure 2
figure 2

PFS with ibrutinib by cytogenetics (FISH)/mutational association. (a) PFS in patients by del17p/del11q CLL subgroups; the del17p subgroup contains patients with del17p with or without del11q (P=0.2160 comparing all three groups). (b) PFS in patients by del17p/TP53 CLL subgroups (P=0.1737 for both del17p and TP53 mutation vs either del17p or TP53 mutation; P=0.0381 for both del17p and TP53 mutation vs neither; P=0.5022 for either del17p or TP53 mutation vs no del17p or TP53 mutation).

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Figure 3
figure 3

PFS with ibrutinib by del17p/TP53 status and complex karyotype. (a) PFS with ibrutinib in patients by any del17p/TP53 alteration vs none. (b) PFS with ibrutinib in all patients with or without complex karyotype.

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A limited multivariate Cox proportional hazards regression analysis (MVA) was performed including the following candidate factors: age, Rai stage, ECOG, number of prior therapies, del11q, del17p, β2-microglobulin, and disease refractory to purine analogues. Gene mutations (for example, TP53 alone) and CK were not included in the MVA due to missing data in ~23% of patients in the ibrutinib and ofatumumab arms. Del17p, del11q and β2-microglobulin were considered significant prognostic factors for PFS in all patients. No significant prognostic factors were identified with current follow-up when the MVA was limited to the ibrutinib arm.

Eighty-six percent of patients randomized to ibrutinib were alive at the time of analysis. Of patients randomized to ofatumumab, 77% were alive with the majority of these patients (108/196) continuing crossover therapy with ibrutinib. Crossover was instituted ~4 months after the last patient was randomized, and OS analyses showed consistent benefit with ibrutinib in the naïve intent-to-treat comparison and when OS was adjusted for crossover (based on rank preserving structural failure time model methodology), with a significant reduction in the risk of death of approximately 64% for ibrutinib vs ofatumumab (Supplementary Figure 3). OS was similar between ibrutinib subgroups including del17p without del11q, del11q without del17p, neither deletion (Supplementary Figure 4) and del17p and/or TP53 (Figure 4a for any alteration vs none, log-rank P=0.1903). No significant difference in OS benefit was observed for ibrutinib-treated patients with respect to known CK, although data were limited (P=0.1610; Figure 4b). The OS 18-month point estimate was 86% in the ibrutinib arm: 83% for the del17p subgroup, 89% for the del11q subgroup, 85% for those with neither deletion, and 79% for those with CK. There was a trend towards improved OS for patients on the ibrutinib arm treated in a second-line setting (HR 2.874, P=0.1324) compared with those salvaged in later lines of therapy (Supplementary Figure 5).

Figure 4
figure 4

Overall survival with ibrutinib by del17p/TP53 status and complex karyotype. (a) Overall survival with ibrutinib in patients by any del17p/TP53 alteration vs none. (b) Overall survival with ibrutinib in all patients with or without complex karyotype.

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Lymphocytosis occurred in 69% of ibrutinib-treated patients and was typically transient, resolving with continued ibrutinib therapy in 87% of patients with a median duration of 14.1 weeks. PFS appeared better for those who developed lymphocytosis vs those who did not (Figure 1d); a previous study has reported non-inferior outcomes between patients with or without persistent lymphocytosis.25 Ibrutinib patients with mutated IGHV showed a slight trend to greater initial increase in absolute lymphocyte count (ALC) compared with those with unmutated IGHV (Supplementary Figure 6), with apparent slower resolution in patients with mutated IGHV vs those with unmutated IGHV similar to a previous report (P<0.001).26

The best ORR increased in the ibrutinib arm with 90% of patients now attaining PR with lymphocytosis (PR-L) or better, as compared with 83 vs 23% (ibrutinib vs ofatumumab, P<0.0001) by investigator assessment at interim analysis.10 ORR was higher in the ibrutinib arm in all evaluable subgroups compared with ofatumumab (P<0.0001). In addition, the complete response (CR) rate improved with 7% of patients demonstrating CR (9 (5%)) or complete response with incomplete blood count recovery (CRi; 4 (2%)) compared with 2% at interim analysis. The latest CR/CRi was achieved in the ibrutinib arm at 17.5 months. One patient on ofatumumab achieved CR at 8 months. The ORR was 100% in ibrutinib-treated patients with only one prior therapy as compared with 88% with ⩾2 prior therapies (P=0.0275, Table 2). No difference was seen in ORR within the ibrutinib arm among patients with or without various genetic mutations. On the ofatumumab arm, patients with del11q and CK had a significantly lower ORR compared with those without del11q and CK (P=0.0038 and P=0.0015, respectively) (Table 2).

Twenty-eight ibrutinib patients progressed including patients who discontinued due to AE and later progressed. Of the 28 patients who progressed, 20 had CLL progression (without RT) and 8 had RT, of which 6 developed large cell lymphoma and 2 Hodgkin disease (5 ofatumumab patients progressed with RT, including 1 after crossover to ibrutinib). Clonal relationship of the RT to prior CLL was unknown. Of the 8 ibrutinib-treated patients with RT (6 within 1 year, 2 within two years), baseline mutations were available for 6 patients and reported as follows: NOTCH1 (n=2), BIRC3 (n=1), TP53 (n=5), SF3B1 (n=1), or ATM (n=1). Other high-risk features were observed in these RT patients, including del17p (n=4), del11q (n=2), and CK (n=3), and in non-RT PD patients (n=20) including del17p (n=9), del11q (n=4), CK (n=5), NOTCH1 (n=7), TP53 (n=11), SF3B1 (n=10). At the time of analysis, 12 of 28 patients who progressed had died (including 8 patients with non-RT progression and 4 with RT). Seventeen of 28 patients had received subsequent anticancer therapy (including 11 patients with non-RT progression and 6 with RT). The most common subsequent therapies were regimens containing ofatumumab (n=6), R-CHOP (n=4) or R±EPOCH (n=4). Of 14 patients who discontinued ibrutinib due to PD and received subsequent anticancer therapy, 6 had RT: 2 died, and 3 were alive at time of analysis on subsequent therapy (See Supplementary Table 3 for details). Eight with non-RT PD received subsequent anticancer therapy, and 3 were alive at analysis.

Safety

The longer-term safety profile of ibrutinib in this randomized phase 3 study was consistent with the 3-year follow-up from the phase 2 study.27 The most common cumulative AE of any grade remains diarrhea, now 54 from 48% at interim analysis, followed by fatigue (34 vs 28%), nausea (31 vs 26%) and fever (30 vs 24%) (Table 3). The most common (>10%) infections included upper respiratory tract infections (25% any grade, 0.5% grade ⩾3), sinusitis (19% any grade, 0.5% grade ⩾3), pneumonia (17% any grade, 12% grade ⩾3), and urinary tract infections (14% any grade, 4% grade ⩾3). Pneumocystis jirovecii pneumonia (grade 3) occurred in 1 patient (0.5%). Seven patients (4%) had grade 5 infections, which included pneumonia (n=4), sepsis (n=2) and neutropenic sepsis (n=1) (Supplementary Table 1); none of these grade 5 infections were considered related to study treatment. Nineteen patients (10%) in the ibrutinib arm had received prophylactic growth factor support, and 131 patients (67%) had received some form of anti-infective prophylaxis, most commonly with antiviral agents including aciclovir (n=53; 27%) and valaciclovir (n=52; 27%). The most common antibiotic agents used for prophylaxis included trimethoprim-sulfamethoxazole (n=47; 24%) and levofloxacin (n=16; 8%).

Table 3 Most common cumulative AEs for ibrutinib (⩾15%) at interim analysis (IA) and current data cut
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New onset of diarrhea, atrial fibrillation (AF), arthralgia and grade ⩾3 infection decreased over time (Table 4). Grade ⩾3 bleeding events were infrequent with three additional events over approximately 1-year follow-up since interim analysis, including grade 3 epistaxis and spontaneous psoas hematoma (the latter during concomitant enoxaparin and clopidogrel) and grade 4 subdural hematoma (post-traumatic event, resolved and patient restarted and continues ibrutinib). Any grade AF occurred in 13 (7%) patients (grade 1 (n=2), grade 2 (n=4), grade 3 (n=7)) with the median time to onset of first event being 5.1 months). Eight (62%) of these patients resolved their AF event (median 3 days to resolution, range 1–42), and all received thromboprophylaxis that included aspirin, low molecular weight heparin, direct oral anticoagulants or combinations thereof (Supplementary Table 4). One patient with AF had a major hemorrhagic event that was the grade 3 spontaneous psoas hematoma described above.

Table 4 Adverse events of interest for ibrutinib-treated patients by time of event onset
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Discussion

Efficacy with ibrutinib remained high at this 2-year follow-up of the RESONATE study, with 74% of patients alive and progression-free. Patients on the ibrutinib arm treated after only one prior therapy experienced improved efficacy compared with those salvaged after ⩾second-line therapy. Phase 3 data in treatment-naive patients without del17p also suggest high efficacy in the frontline setting with significantly improved PFS and OS compared with chlorambucil with a median 19-month follow-up.28 Taken together, the results from this study as well as from studies of ibrutinib in frontline therapy27, 28 suggest that the best results occur when ibrutinib has been used earlier in the treatment course. This observation has also been true of other therapies historically, including chemoimmunotherapy with FCR, perhaps reflecting the accumulation of multiple and broad mechanisms of drug resistance over time with treatment. The outcome of ongoing randomized trials using standard combination chemoimmunotherapy vs ibrutinib-containing regimens in the upfront setting will be required to determine the true extent of ibrutinib benefit in that setting as compared with the early relapsed setting.

Tolerability to ibrutinib was well maintained in this relapsed patient population with extended treatment. The most common side effects were similar to those at interim analysis with only a slight increase in the cumulative event rate despite the additional year of therapy. Specifically, the rate of new infection, diarrhea and arthralgia appeared lower later in follow-up when compared with the first 6 months, consistent with a recent report of long-term follow-up with ibrutinib at the Ohio State University, in which patients who discontinued for reasons other than progression did so relatively early, followed by a plateau.29 Severe (grade ⩾3) bleeding events were infrequent with three events occurring during the additional follow-up period, with two of these events occurring in the context of concomitant anticoagulation and in a post-traumatic setting, respectively. Recent studies have reported reduced ristocetin-30and collagen-31 mediated platelet aggregation in patients treated with ibrutinib. Grade ⩾3 bleeding events with ibrutinib were uncommon in these reports. With regard to AF, the median time of onset of 5.1 months in our study was similar to 3.8 months reported in the largest cohort of ibrutinib-treated CLL patients with AF (n=56).32 Further, hypertension has been noted throughout the ibrutinib treatment course including at later times.27, 28 Thus, ongoing monitoring of toxicity, particularly in patients who may remain on ibrutinib for many years, remains important.

Prior non-randomized trials have reported that RT tends to occur early in the course of ibrutinib treatment29, 33, 34 followed by a plateau in incidence. In this study, which followed patients only until disease progression, more events of RT were reported in the ibrutinib arm compared with the ofatumumab arm (8 patients vs 5 patients, respectively, with 6 and 4 patients with RT in the first year of follow-up), but these frequencies reflect a median PFS (and therefore of follow-up) of 8.1 months on ofatumumab vs ongoing follow-up in most patients on ibrutinib. Previous reports of three randomized controlled CLL trials have identified a similar incidence of RT on the ibrutinib and control arms; there were 3 patients with RT on the placebo arm vs 0 on the ibrutinib arm of HELIOS,35 1 RT on the chlorambucil arm vs 0 on the ibrutinib arm of RESONATE-2,28 and 2 RT cases on each arm of RESONATE at time of primary analysis.10 Moreover, the 4% rate of RT in the ibrutinib arm of our current analysis is not different from historical rates reported with other CLL regimens.36, 37, 38 Patients who developed RT in the ibrutinib arm had frequent adverse genomic features at baseline, including del17p, NOTCH1, and TP53 abnormalities, which are known to be associated with, and may contribute to, the development of RT. Thus, the risk of RT with ibrutinib treatment does not appear to be higher compared with other treatment regimens in CLL, and likely reflects the natural history of high-risk, relapsed/refractory disease.

For the first time, we report randomized efficacy outcomes with ibrutinib in high-risk FISH-defined genetic subgroups in addition to more novel high-risk gene mutations. Ibrutinib markedly improved PFS and ORR in all genetic subgroups over ofatumumab. In particular, with an additional year of follow-up, patients on ibrutinib with either del17p or TP53 mutation did not show markedly worse PFS than those without these genetic abnormalities, although in an exploratory analysis, the presence of both abnormalities was associated with a decrease in the PFS curve. Notably, similar to prior reports,39 >1/2 of patients with TP53 mutations did not have del17p – such patients experience poor outcomes to chemotherapy regimens,40 yet TP53 mutational testing is not standardly performed in the United States. The 3-year update of the phase 2 data demonstrated that patients with del17p have significantly lower PFS (median 28 months) than patients with del11q or neither of these anomalies, where the median PFS was NR.27 In that study, TP53 mutation was not tested, but it is likely that most patients with del17p had TP53 mutation, as previously reported;24 thus, these findings are likely consistent with the emerging decrease in PFS observed in the current study for patients with both abnormalities. In a subsequent efficacy analysis of patients with del17p from the same study, patients without CK appeared to experience the most favorable PFS/OS outcomes.41 Consistent with these findings, investigators at MD Anderson Cancer Center and Ohio State University have previously reported that CK adversely affects ibrutinib outcomes.29, 42 This finding was not observed yet in this study, but may be due to incomplete data or still relatively short follow-up.

Analysis of other relevant prognostic genes including SF3B1, BIRC3, ATM and NOTCH1 revealed no difference in progression outcomes between ibrutinib patients with or without these mutations at 19 months follow-up. In contrast, NOTCH1-mutated patients randomized to ofatumumab fared significantly worse than their non-mutated counterparts, perhaps consistent with previous reports in which NOTCH1 mutations were associated with lack of benefit of CD20 antibody therapies.18, 19 It should be noted that, in order to make our results relevant to clinical practice, we used a clinically available next generation sequencing panel applied only to tumor, and because of that, the analysis may be limited by the absence of comparison to matched germline, which may result in some rare germline variants being counted as somatic mutations. In addition, all mutations detected in the NGS panel, which has a detection limit of approximately 1% allele frequency, were included in the clinical analysis, yet at present, it is unknown whether a cutoff should be applied for a minimum allele frequency when assessing these mutations in relation to clinical outcome. Given this uncertainty, we chose to err on the side of including any detected candidate mutation, particularly as prior literature does suggest that even tiny TP53 mutated clones are associated with poor outcome.39, 43 Ultimately, detailed scientific studies with large uniformly treated datasets will be required to determine whether an allele frequency cutoff would be more appropriate.

In this study, patients who developed lymphocytosis experienced longer PFS compared with those who did not. In a 3-year follow-up of treatment-naïve and relapsed/refractory patients with CLL/SLL, Byrd et al. reported similar PFS outcomes in patients with and without persistent lymphocytosis at one year of ibrutinib therapy.27 Woyach et al. have also reported similar outcomes among patients with or without persistent lymphocytosis treated with ibrutinib, although a trend towards improved PFS was seen in patients who achieved PR-L.25 An association between prolonged lymphocytosis and favorable prognostic features was also observed, with ibrutinib responders with prolonged treatment-related lymphocytosis more likely to carry favorable prognostic markers including del13q and mutated IGHV.25 Taken together, these data suggest that prolonged lymphocytosis is associated with favorable prognostic factors that are associated with longer response, but ultimately longer follow-up is warranted to establish a definite correlation.

Ultimately, given the low number of PFS events in patients treated with ibrutinib, the follow-up is still too short to definitively know whether subgroups characterized by higher-risk mutations, including del17p, will have reduced PFS. Meanwhile, the majority of relapsed CLL patients treated on this trial continue to do extremely well, underscoring the significant impact of ibrutinib in altering the course of relapsed CLL.