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Ibrutinib efficacy and tolerability in patients with relapsed chronic lymphocytic leukemia following allogeneic HCT

Christine E. Ryan, Bita Sahaf, Aaron C. Logan, Susan O’Brien, John C. Byrd, Peter Hillmen, Jennifer R. Brown, Martin J. S. Dyer, Anthony R. Mato, Michael J. Keating, Samantha Jaglowski, Fong Clow, Andrew R. Rezvani, Lori Styles, Steven E. Coutre and David B. Miklos

Key Points

  • Ibrutinib provided effective salvage therapy in CLL relapse post–alloHCT, resulting in sustained MRD negativity without GVHD development.

  • Ibrutinib selectively depleted pre–germinal B cells and Th2 helper cells and may enhance donor Th1 T-cell–mediated GVL effects.

Abstract

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton’s tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism–associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell–mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre–germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.

Introduction

Allogeneic hematopoietic cell transplant (allo-HCT) is used to treat patients with high-risk chronic lymphocytic leukemia (CLL) and provides long-term disease-free survival.1 In a recent retrospective study of 52 CLL patients who progressed following HCT, the 2- and 5-year overall survival rates posttransplant were 67% and 38%, respectively.2 These results demonstrate the continued need for additional effective therapeutic agents in the setting of post–allo-HCT relapse.

Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton’s tyrosine kinase and interleukin-2 inducible kinase (ITK), as well as several other tyrosine kinases, has been used to treat relapsed/refractory (R/R) CLL, with high response rates and prolonged progression-free and overall survival.3-8 In one study of 132 CLL patients with either treatment-naive or R/R CLL, the most common responses to single agent ibrutinib were durable partial responses, with 83% overall survival rate at 30 months of follow-up.9 ITK plays a key role in the activation of both Th1 and Th2 cells. In Th1 cells, ITK signaling is supportive but dispensable to redundant resting lymphocyte kinase signaling, whereas in Th2 cells, ITK signaling is essential to activation.10-12 The subversion of the Th2 subpopulation via ibrutinib results in skewing away from a Th2 cytokine profile, as evidenced by the observed decrease in interleukin 10 (IL-10), IL-4, and IL-13, in patients with CLL treated with ibrutinib.12

The inhibition of ITK is of unique interest following allogeneic transplant because we postulate that the Th1-mediated graft-versus-leukemia (GVL) benefit will reduce leukemia relapse risk. Recent studies have reported on small numbers of patients with CLL who relapsed following allo-HCT and were treated with ibrutinib as a salvage therapy with promising results.2,13 Here, we present 27 patients with relapsed CLL following allo-HCT who benefited from ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials, and an additional 11 patients were treated at Stanford University following US Food and Drug Administration (FDA) approval of ibrutinib. In our studies, ibrutinib proved to be a safe and effective salvage therapy for CLL following allogeneic transplant. Ibrutinib led to sustained disease response and therapeutic donor immune modulation, improving GVL effects without contributing to graft-versus-host-disease (GVHD) development. Our comprehensive immune phenotype characterization of peripheral B and T cells collected from Stanford patients treated with ibrutinib shows that ibrutinib selectively targets pre–germinal center B cells and depletes Th2 helper cells. Furthermore, these immune modulatory effects persist following treatment discontinuation.

Patients and methods

Multi-institutional clinical trial cohort

Data were first collected for patients with R/R CLL who had undergone prior allo-HCT and had been treated with ibrutinib on a Pharmacyclics-sponsored clinical trial, either as a single agent or in combination with ofatumumab. These patients were in 1 of 4 clinical trials (supplemental Table 1, available on the Blood Web site). All patients received 420 mg ibrutinib daily, except for 2 patients who received 840 mg daily. Efficacy evaluation included response assessment according to the International Workshop on CLL criteria (2008), and hematologic adverse events (AEs) were graded using the Hallek criteria.14

Stanford University cohort

We additionally studied 11 Stanford allo-HCT patients who experienced CLL relapse as defined by the 2008 International Workshop on CLL criteria14 after FDA approval of ibrutinib in November 2013 and were not already part of the aforementioned clinical trials. These patients received monotherapy with 420 mg ibrutinib daily and were monitored on an institutional review board–approved research blood sample protocol. They were evaluated for response at 1 month and 3 months and at subsequent 3-month intervals, with concurrent AE evaluation.14 The cutoff date for data assessment was 1 March, 2016.

MRD monitoring and donor chimerism assessment

Minimal residual disease (MRD) and donor CD3 chimerism analysis15 was performed for all patients treated at Stanford. This included the 11 patients treated off-trial and 2 of the multi-institutional trial cohort patients. CLL MRD was measured by immunoglobulin H (IgH) high-throughput sequencing using the ClonoSeq test (Adaptive Biotechnologies, Seattle, WA).16,17

Immune phenotype analysis

Multiparameter flow cytometric analysis was carried out with cell staining methods as described previously.18,19 After thawing cryopreserved peripheral blood mononuclear cell (PBMC) samples, cell suspensions were tested with the LIVE/DEAD kit (Life Technologies, San Diego, CA) with at least 85% viability after thawing. Cells were then washed and stained for 15 minutes with mixtures of fluorochrome-conjugated antibodies against cell-surface marker proteins, including CD3, CD4, CD5, CD8, CD19, CD23, CD24, CD25, CD27, CD38, CD45, CD45RA, CD45RO, CD62L, CCR7, CD127, CXCR5 (CD185), IgD, IgM, Vα24, and CD230. The 21 antibodies were measured in multiplex fashion, with antibodies segregated by fluorochrome labels into 4 separate incubation mixtures. A fifth incubation mixture was used to assess the levels of the intracellular proteins T-bet, GATA3, and BCL-6. Following cell-surface staining, PBMCs were washed and resuspended in 4% formaldehyde solution for 2 to 16 hours at 4°C. Fixed PBMCs were then incubated in permeabilization buffer (BioLegend, San Diego, CA) and further incubated for 30 minutes with antibodies against the intracellular proteins. All aforementioned antibodies were obtained from BioLegend, except for the Vα24 antibody, which was obtained from eBioscience (San Diego, CA). Flow cytometry data were acquired using an LSRII flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ) and analyzed using FlowJo (FlowJo, Ashland, Oregon).

Results

CLL trial cohort patient characteristics

A total of 16 patients from 4 ibrutinib clinical trials for relapsed CLL had undergone prior allo-HCT (Table 1; supplemental Table 1). Median patient age was 54.5 years (range, 43-68), and 11 patients were male. Ten of 16 patients had a del17p chromosomal abnormality, and 3 patients had a del11q chromosomal abnormality. Twelve of the 16 patients had undergone 4 or more therapies prior to ibrutinib initiation. Ibrutinib was initiated at a median time of 27 months posttransplant (range, 8-115).

Table 1.

Summary of patient characteristics at time of ibrutinib initiation

Stanford Cohort patient characteristics

The Stanford cohort not treated as part of the trials described above consisted of 11 patients (Table 1; supplemental Table 2). They relapsed between 1 month and 8.5 years following reduced-intensity conditioning allo-HCT with leukemic clonal progression quantified by ClonoSeq. Ten of the 11 patients had pathological lymphadenopathy progression. Biopsies confirmed CLL relapse, and Epstein-Barr virus stains of biopsy samples were negative. Prior to the initiation of ibrutinib, all patients failed to respond to immune suppression taper and, in several cases, chemotherapy and/or donor lymphocyte infusion (DLI).

Median patient age at time of ibrutinib initiation was 59.4 years (range, 41-68), and 7 of 11 patients were male. Four patients had a del17p chromosomal abnormality, and 4 patients had a del11q chromosomal abnormality. Nine of the patients had undergone 4 or more therapies prior to ibrutinib initiation. Ibrutinib was initiated at a median time of 73.4 months posttransplant (range, 2.1-100.7).

CLL disease response and ibrutinib tolerability: CLL trial patients

In the cohort of patients treated as part of multi-institutional clinical trials, 15 patients with evaluable lymphadenopathy experienced dramatic reduction in lymph node size, with an overall median reduction of 88.6% (Figure 1A). In this cohort, there was an overall response rate (ORR) of 87.5% (14 of 16 patients), as determined by investigator-assessed responses (Figure 1B). None of the investigator-assessed best responses were progressive disease. Two patients achieved a complete response, 10 patients achieved a partial response, and 2 patients had partial responses with lymphocytosis. One patient had stable disease, and 1 patient with a del17p chromosomal abnormality was not evaluable for response. The ORR response rate was similar between all patients assessed and the subset of patients with a del17p chromosomal abnormality, in which the ORR was 80%. The 24-month progression-free survival and overall survival rates were each 77% (Figure 1C) and 75%, respectively.

Figure 1.

Outcomes in post–allo-HCT relapsed CLL patients treated with ibrutinib. (A) Lymph node response for 15 patients shown as the percent change from baseline, measured by the sum of the products of the lymph node diameters (SPD). One patient was not evaluable for response. Median percent reduction was 88.6%. (B) Investigator-assessed response rates. One patient with a del17p chromosomal abnormality was not evaluable for response. No patients on the study showed disease progression as best response; 1 patient in each group did not have a postbaseline response assessment. (C) Kaplan-Meier curve showing progression-free survival of 77% at 24 months postibrutinib initiation. (D) The most common treatment-emergent AEs (occurring in ≥15% of patients) are shown for this cohort of patients treated as part of the multi-institutional clinical trials. CR, complete response; PR, partial response; PR-L, partial response with lymphocytosis; SD, stable disease.

In this cohort, median duration of ibrutinib treatment was 19 months (range, 0.4-39), and 14 patients were treated with ibrutinib for longer than 12 months. No dose reductions were reported. At the time of data cutoff, 11 patients were continuing treatment. Reasons for discontinuation included disease progression (n = 2), AEs (n = 2), and withdrawal of consent (n = 1).

The most common treatment-emergent AEs observed in these clinical studies were diarrhea (n = 11), upper respiratory tract infection (n = 5), cough (n = 5), contusion (n = 5), and arthralgia (n = 5) (Figure 1D; supplemental Table 3). Treatment-emergent grade ≥3 severe AEs were observed in 12 patients (supplemental Table 4). These severe AEs included pneumonia (n = 2), 3 serious bleeding events (subdural hematoma [n = 1], perirenal hematoma [n = 1], postprocedural hemorrhage [n = 1]), and the following additional events (n = 1 for each event): atrial flutter, bone lesion, bronchitis, Campylobacter gastroenteritis, colitis, dyspnea, febrile neutropenia, neutropenia, hematuria, herpes zoster, herpes zoster disseminated, hypercalcemia, lymph gland infection, otitis externa, otitis media, influenza pneumonia, organizing pneumonia, syncope, and urinary retention. The only AE leading to discontinuation of ibrutinib was pneumonia in 2 patients; both were fatal events (at 0.9 and 2.3 months). Two additional deaths occurred on study due to disease progression at 24 and 28 months.

CLL disease response and ibrutinib tolerability: Stanford cohort

In the 11 patients treated as part of the Stanford off-trial cohort, the ORR was 91%. Seven patients (64%) achieved a complete response, 3 patients (27%) achieved a partial response, and 1 patient (9%) who had initial response later developed progressive disease (supplemental Table 2). The one patient who developed progressive disease (SPN5435) died after 8 months of therapy. The higher ORR observed in the Stanford cohort is likely due to a lower disease burden at time of ibrutinib initiation, which was more proximal to post–allo-HCT relapse, as ibrutinib had already been FDA approved.

The median duration of ibrutinib treatment in this cohort was 8.4 months (range, 0.5-21.4) with 6 patients continuing treatment. One patient (SPN3873) discontinued ibrutinib due to drug intolerance manifesting as a papular rash with pruritus and oral ulcers. All of these symptoms resolved within 7 days of drug discontinuation, and the patient did not have GVHD. Two additional patients died while on ibrutinib despite achieving clinical benefit. One patient (SPN5919) had active human herpesvirus 6 viremia at time of graft infusion and succumbed to uncontrollable human herpesvirus 6 encephalitis (at 0.5 months of treatment, 3 months posttransplant). The second patient (SPN5904) experienced sudden death (at 1.5 months of treatment, 5 months posttransplant); autopsy showed pericarditis and right atrial thrombus and, notably, no lymphadenopathy. Other AEs experienced in this patient cohort included fatigue (n = 7), diarrhea (n = 1), nausea (n = 1), and pericardial effusion (n = 1).

Lymph node reduction

For the patients treated at Stanford with lymphadenopathy and available computed tomography scan assessment (n = 8), there was an average 73% reduction in lymph node size after only 6 months of treatment (supplemental Figure 1). One patient (SPN3431) presented at relapse with a pelvic mass confirmed by core needle biopsy to be CLL. The mass measured 16 cm by 13 cm and was eroding the pelvic bone and compressing the patient’s vagina and uterus. Remarkably, within 7 months of ibrutinib treatment, she had undetectable CLL levels by IgH high-throughput sequencing, and a computed tomography scan at 9 months of ibrutinib treatment showed almost complete resolution of the pelvic mass (supplemental Figure 2).

Ibrutinib post–allo-HCT achieves MRD negativity

Blood CLL burden was followed in 11 patients by ClonoSeq (supplemental Table 2). Ibrutinib therapy achieved CLL MRD negativity (<1 CLL clone per 10 000 white blood cells [WBCs]) in 4 patients (36%), with 3 of these patients achieving undetectable levels of CLL (<1 CLL clone per 1 million WBCs; Figure 2A-B,F). Ibrutinib initiation was associated with a transient 1-log increase in peripheral blood CLL with subsequent decrement (supplemental Figure 3). This initial increase, most prominent in Figure 2B, is attributed to the mobilization of CLL cells into the peripheral blood as previously described.20,21

Figure 2.

Ibrutinib treatment leads to decreased CLL levels and increased donor chimerism levels. CLL clones per 1 million WBCs and blood donor CD3 T-cell percentage were assessed at various time points posttransplant and during ibrutinib treatment. Data are shown for 6 representative patients: (A) SPN3975, (B) SPN3431, (C) SPN4158, (D) SPN3873, (E) SPN3697, and (F) SPN3903.

Remarkably, MRD negativity persisted following the discontinuation of ibrutinib. Two patients (SPN3975 and SPN3431; supplemental Table 2) discontinued ibrutinib after their CLL levels became undetectable, and both continue to have MRD <0.01% up to 9 months and 26 months, respectively, after discontinuation (Figure 2A-B). In addition, patient SPN3873, who stopped ibrutinib due to drug intolerance, continued to have decreasing CLL burden off ibrutinib and achieved MRD negativity 18 months after discontinuation (Figure 2D).

Favorable effects on donor chimerism

Donor chimerism levels were followed for all patients treated at Stanford. CLL progression following allo-HCT is associated with mixed donor T-cell chimerism,22,23 and indeed, 9 patients treated at Stanford had mixed chimerism–associated CLL relapse (range of CD3 donor chimerism at relapse, 8% to 95%). With ibrutinib initiation, 4 of these patients (44%) converted from mixed to full donor chimerism (shown in Figure 2A,C-E). Conversion required 5 to 11 months of therapy. Specifically, SPN3975 had failed to maintain full donor CD3 chimerism after rituximab and 5 dose-escalated donor lymphocyte infusions (dose range, 1 × 107 to 1 × 108 CD3+ cells per kilogram recipient weight) but after 1 year of ibrutinib treatment achieved full donor chimerism (Figure 2A). Four patients relapsed following allo-HCT despite having full donor chimerism.

No GVHD development

None of the 27 patients developed GVHD following initiation of ibrutinib treatment. One patient who had developed oral and skin chronic GVHD (cGVHD) following donor lymphocyte infusions experienced full resolution of these cGVHD symptoms after 6 months of ibrutinib treatment (Figure 2A).

Ibrutinib selectively targets pre–germinal center B cells and depletes Th2 helper cells

We conducted blood immune phenotype characterization of PBMC samples from 10 patients who were treated at Stanford; samples were from 1, 3, 6, and 12 months following ibrutinib initiation. Multiparameter flow cytometric analysis for the B- and T-cell populations studied are shown in Figures 3A and 4A, respectively. Analysis of the subsets of CD19+ B-cell populations shows that ibrutinib rapidly depleted CLL (CD5+CD23+) following an initial brief peripheral increase (Figure 3B; supplemental Table 5). Ibrutinib also depleted the CD19+CD38+CD27+IgD+ cells, previously described as pre–germinal center (pre-GC) B cells, which have been shown to associate with cGVHD,24 with a statistically significant decreased pre- to postibrutinib ratio after only 3 months of ibrutinib treatment (P < .01). In contrast, CD19+CD27+IgDCD38 isotype-switched memory B cells persisted throughout ibrutinib therapy (Figure 3B).

Figure 3.

Immune characterization of peripheral B cells from patients following initiation and discontinuation of ibrutinib. Multiparameter flow-cytometric analysis was completed with B cells from PBMC samples from 10 patients with relapsed CLL post–allo-HCT treated with ibrutinib at Stanford. Data are shown for 3 clinically relevant B-cell subsets: CLL, memory B cells, and pre-GC–activated B cells. (A) Blood samples from a representative patient that were collected preibrutinib and 3 months following ibrutinib initiation were analyzed by sequential gating as shown. All of the patients’ samples were analyzed with the same sequential gating depicted. Green, blue, and red gates mark the various cell populations determined to be positive compared with an FMO (fluorescence minus 1). These colors correspond to the line graphs in panels B and C, as well as to the absolute cell numbers listed in supplemental Table 5. (B) The change following ibrutinib initiation for each clinically relevant B-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with standard error of the mean (SEM) (n = 10 at preibrutinib, and n = 5 at 1, 3, 6, and 12 months postibrutinib initiation). (C) The change following ibrutinib discontinuation for each clinically relevant B-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with SEM (n = 3 at all time points).

Figure 4.

Immune characterization of peripheral T cells from patients following initiation and discontinuation of ibrutinib. Multiparameter flow-cytometric analysis was completed with T cells from PBMC samples from 10 patients with relapsed CLL post–allo-HCT treated with ibrutinib at Stanford. Data are shown for 4 clinically relevant T-cell subsets: total CD4+, Th1 cells, Th2 cells, and CD8+ cells. (A) Blood samples from a representative patient that were collected preibrutinib and 3 months following ibrutinib initiation were analyzed by sequential gating as shown. All of the patients’ samples were analyzed with the same sequential gating depicted. Gray, blue, red, and black gates mark the various cell populations determined to be positive compared with an FMO (fluorescence minus 1). These colors correspond to the line graphs in panels B and C, as well as to the absolute cell numbers listed in supplemental Table 6. (B) The change following ibrutinib initiation for each clinically relevant T-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with SEM (n = 10 at preibrutinib and n = 5 at 1, 3, 6, and 12 months postibrutinib initiation). (C) The change following ibrutinib discontinuation for each clinically relevant T-cell subset is graphed as the ratio of the absolute number of cells detected postibrutinib to the absolute number of cells in the same subset preibrutinib. Ratios are plotted as mean with SEM (n = 3 at all time points).

Analysis of the T-cell subpopulations showed that the absolute number of CD3+CD8+ T cells was unchanged at an average of 490 cells/μL at 6 months of ibrutinib treatment (Figure 4B; supplemental Table 6). In contrast, CD4+ T cells were 50% reduced from an average of 430/μL preibrutinib to an average of 210/μL by 6 months. Further analysis of the CD4+ T cells shows a 75% reduction of GATA3 expressing Th2 CD4+ cells, while in contrast, there was no significant change in the number of Tbet-expressing Th1 CD4+ cells. In addition, initiation of ibrutinib caused a rapid depletion of CD4+CXCR5+BCL6+ follicular helper T (TFH) cells from circulation, with none detected by 3 months (P < .01) (Figure 4B).

Immune modulatory effects of ibrutinib persist after treatment discontinuation

We conducted the same immune phenotype assessment of PBMC samples from 3 patients who discontinued ibrutinib; samples were from 3, 6, and 12 months after ibrutinib discontinuation. These results revealed that compared with preibrutinib treatment, the CD19+CD38+CD27+IgD+ pre-GC subpopulation remained depleted while the CD19+CD27+IgDCD38 memory B cell subpopulation persisted following ibrutinib discontinuation (Figure 3C). Analysis of the T-cell subpopulations after ibrutinib discontinuation revealed that compared with preibrutinib samples, levels of GATA3 expressing Th2 CD4+ cells remained unchanged, while in contrast, levels of Tbet-expressing Th1 CD4+ cells were relatively increased (Figure 4C).

Discussion

In total, ibrutinib provided effective and tolerable salvage therapy for CLL relapse following allogeneic HCT. Here, we present 27 patients who were treated with ibrutinib after CLL relapse following allo-HCT. The large majority (81%) of the patients in the clinical trial cohort had failed intervening salvage therapies posttransplant, including rituximab, combination chemotherapy, and DLI. Despite these previous treatment failures, ibrutinib treatment provided an 87.5% ORR. Furthermore, this response was persistent, with only 3 additional progressions occurring after the 24-month observation period. In the Stanford cohort, there was similarly a 91% ORR. Our results contrast those recently reported by Rozovoski et al in which the 2- and 5-year survival rates for patients with CLL disease progression following allo-HCT were only 67% and 38%, respectively, demonstrating that the majority of patients with progressive disease posttransplant have poor long-term survival.2 They presented 5 patients treated with ibrutinib who all had clinical responses and were alive after a median follow-up duration of 16 months. Similarly Link et al recently reported that 5 post–allo-HCT CLL patients treated with ibrutinib salvage therapy achieved partial responses with no relapse at 1 year of follow-up. One patient achieved MRD negativity.13

Ibrutinib was tolerated with a safety profile similar to the one observed in the overall population of patients with R/R CLL treated with ibrutinib in the phase 3 clinical trial as part of the RESONATE study. In that cohort, 57% of patients in the ibrutinib group had at least 1 AE of grade 3 or higher.8 In our multi-institutional cohort of trial patients who had undergone allo-HCT prior to ibrutinib initiation, 69% of patients had at least 1 AE grade 3 or higher (11 of 16 patients). The most common AEs in this cohort of trial patients post–allo-HCT were diarrhea (69%), upper respiratory tract infection (31%), cough (31%), arthralgia (31%), and contusion (31%). In the patients treated with ibrutinib in the RESONATE study, these were also some of the most common AEs, occurring with similar frequency. Diarrhea occurred in 48% of patients, cough in 19%, upper respiratory tract infection in 16%, and arthralgia in 17%.8 Causes of ibrutinib discontinuation in the Stanford off-trial cohort are similar to those listed in a report on patients with R/R CLL treated with ibrutinib as part of the phase 2 or 3 clinical trials who then discontinued ibrutinib. In that study, reported causes included disease progression, a case of recurrent ulcers, and 2 cases of sudden death.25 Overall, in comparison with alternative therapies, the AEs are similar to, if not better than, those that would be expected for those with combination chemotherapy, DLI, or even repeat transplant.

Our studies were initiated after the remarkable outcome of SPN3975, the sixth patient to ever receive ibrutinib on the first clinical trial testing ibrutinib (PCYC1102). Despite failure of multiple intervening CLL therapies following relapse 1 year posttransplant (including oxaliplatin/cytarabine/fludarabine/rituximab,26 dose-escalated rituximab, and dose-escalated DLI), it was ibrutinib treatment that successfully led to conversion of the T-cell compartment to full donor chimerism and achieved undetectable CLL levels (Figure 2A).27 In this index patient, we see 3 key effects of ibrutinib treatment in the post–allo-HCT relapse setting, which are evident in our cohort of Stanford patients. First, patients treated with ibrutinib have achieved complete response in association with conversion to full donor T-cell chimerism conversion (44%). Second, ibrutinib achieved CLL MRD negativity in 4 patients (36%). Third, MRD <0.01% persisted even after ibrutinib was discontinued, in 1 case even after 26 months.

We postulate that ibrutinib is achieving sustained GVL augmentation through a T-cell–mediated effect due to ibrutinib inhibition of ITK activity. Both Th1 and Th2 cells rely on ITK signaling. However, in Th1 cells, resting lymphocyte kinase provides an additional signaling pathway that theoretically rescues the Th1 cells from the ibrutinib-induced ITK inhibition, leading to disproportionate effects on Th2 cells.12 Previous studies investigated the effects of ibrutinib on Th1 and Th2 subpopulations via analysis of cytokines in primary samples of patients treated with ibrutinib. One study reported an observed decrease in serum Th2-type cytokines, including IL-10, IL-4, and IL-13,12 and another similarly showed a decrease in IL-10 levels following treatment.13

Here, we report for the first time comprehensive immune phenotype of B and T cells using primary samples from patients with CLL treated with ibrutinib following allo-HCT. Our results show that ibrutinib leads to dramatic depletion of Th2 cells. This subpopulation is decreased as early as 3 months after ibrutinib initiation and continues to decrease with continued treatment (Figure 3B). On the other hand, the Th1 subpopulation and cytotoxic CD8+ T cells persist without any evidence of depletion. Thus, our results provide evidence that ibrutinib may augment the GVL effect through persistence of the Th1 and cytotoxic CD8+ T-cell populations, resulting in CLL MRD eradication. Ibrutinib efficacy in the post–allo-HCT setting suggestive of augmented GVL effect has previously been reported in 2 patients with refractory classic Hodgkin’s lymphoma.28 A recent study in a MCL mouse xenograft model similarly demonstrated the role of ibrutinib in augmenting antitumor activity. The addition of ibrutinib to anti-CD19 chimeric antigen receptor T cells led to enhanced MCL killing and higher rates of long-term remission.29 A pilot trial is now underway to assess the efficacy of combining ibrutinib with anti-CD19 chimeric antigen receptor T cells to treat R/R CLL (NCT 02640209).

An emerging hypothesis for the mechanism of ibrutinib-mediated GVL benefit is that ibrutinib effectively disrupts the CLL tumor microenvironment and reduces T-cell pseudoexhaustion.30,31 Studies have shown that CD8+ T cells from patients with CLL exhibit a state of acquired dysfunction with defects in proliferation and cytotoxicity.32 This exhaustion is thought to develop from chronic activation, similar to the phenomenon observed in chronic viral infections. Direct contact between T cells and tumor cells from patients with CLL induces defective actin polymerization and subsequent immunological synapse formation, which can impair cytotoxicity.33,34 Recent studies of primary samples from patients with CLL treated with ibrutinib show that ibrutinib may decrease chronic T-cell activation, as evidenced by a decrease in the activation markers HLA-DR and CD39.30 Decreased PD-1 surface expression was also observed, and it has been shown that the combination of ibrutinib and either anti–PD-L1 antibodies or intratumoral CpG injection enhances antitumor responses,35,36 suggesting that increased T-cell activation is critically important. Another recent study investigating the effect of ibrutinib on chimeric antigen receptor–expressing T cells demonstrated that defects in T-cell proliferation and activation were improved after 5 to 11 cycles of ibrutinib therapy.37 Thus, it is possible that direct elimination of CLL through ibrutinib Bruton’s tyrosine kinase targeting reduces T-cell exhaustion, leading to more effective T-cell immunity.

Remarkably, these GVL effects are accomplished in the absence of GVHD. None of the 27 patients treated as part of this study developed GVHD. Many of the therapies given to CLL patients who relapse following allo-HCT, including DLI, lead to an increased risk of GVHD.38 Notably, 1 patient in this study (SPN3975) who had persistent cGVHD prior to ibrutinib initiation experienced full resolution of cGHVD symptoms within 6 months of treatment. In murine studies, ibrutinib has already been shown to improve clinical and pathological manifestations of cGHVD.39

Our multiparameter flow-cytometric analyses provide insight into identifying the possible mechanisms by which ibrutinib affects the process of GVHD development. Ibrutinib targets pre-GC B cells, which have been shown to associate with cGVHD, and causes rapid depletion of CD4+CXCR5+BCL6+ TFH cells from the circulation (Figure 4B). TFH cells play an important role in B-cell activation,40 and much of our group’s previously published work has shown the critical role that B cells, and specifically antibodies to HY antigens, play in the development of GVHD.19,41,42 Recent studies specifically linked TFH cells to the promotion of B cell involvement in cGVHD.43 Depletion of both pre-GC B cells and TFH cells may thus contribute to protection against GVHD development. The efficacy of ibrutinib in treating cGHVD requires further investigation, and a phase 1/2 clinical trial of ibrutinib treatment of patients with cGVHD who have failed therapy with corticosteroids is underway to assess this area of therapeutic potential (NCT #02195869).

Limitations to this study include the heterogeneity of the patient population. All patients had received a wide range of previous therapies and started ibrutinib therapy at various intervals following both allo-HCT and relapse post–allo-HCT. Furthermore, while we have proposed that ibrutinib augments GVL benefit through Th2 subversion and enhancement of cytotoxic CD8+ T-cell activity, we have not interrogated these mechanisms directly. Ongoing studies of mixed-lymphocyte reactions with patient T cells against primary CLL before and after ibrutinib exposure will provide further elucidation.

Although it has been reported that ibrutinib monotherapy for CLL rarely achieves MRD negativity,9,44 4 patients in this study achieved MRD negativity with ibrutinib treatment. The fact that ibrutinib leads to MRD negativity in the post–allo-HCT setting, but not as initial monotherapy pretransplant, suggests that ibrutinib-related enhancement of the GVL effect in the posttransplant setting contributes to the achievement of MRD negativity. In analyzing those patients in our study who achieved MRD negativity versus those who did not, we explored factors that would have an impact on alloimmunity, including preceding use of rituximab or DLI, tapering of immune suppressive medications following ibrutinib initiation, and development of full donor chimerism. However, our patient sample size was unfortunately too small to observe any correlations or make any conclusions regarding which factors may be augmenting the efficacy of ibrutinib in the posttransplant setting. This will be the groundwork for future studies that continue to investigate the mechanisms by which ibrutinib mediates both effective suppression of CLL and Th2 T cells and enhancement of the donor Th1 T-cell–mediated GVL benefit.

Authorship

Contribution: C.E.R., B.S., L.S., and D.B.M. designed research; C.E.R., B.S., A.C.L., S.O., J.C.B., P.H., J.R.B., M.J.S.D., A.R.M., M.J.K., S.J., F.C., A.R.R., L.S., S.E.C., and D.B.M. collected data; B.S., C.E.R., and D.B.M. performed experiments; C.E.R., B.S., and D.B.M. wrote the manuscript; and all authors revised and approved the final version of the manuscript.

Conflict-of-interest disclosure: B.S. holds a patent with Stanford University. A.C.L. receives consultancy and research funding from Pharmacyclics, consultancy funding from Amgen and Jazz, and research funding from Astellas and Novartis. S.O. receives honoraria and consultancy funding for Janssen; honoraria, consulting, and research funding from Pharmacyclics. P.H. receives honoraria, consultancy, and research funding from Roche, GSK, Janssen, Gilead, and AbbVie, as well as honoraria and research funding from Novartis and Pharmacyclics and research funding from Celgene. J.R.B. receives honoraria, consultancy, and travel accommodations from Janssen, Gilead, and Pfizer, as well as honoraria and consultancy funding from Pharmacyclics, Celgene, Infinity, and Roche/Genentech and honoraria and travel accommodations from Sun BioPharma. M.J.S.D. is on the speaker’s bureau and consults for Roche and receives travel accommodations and research funding from Gilead and research funding from Ono. A.R.M. receives consultancy, research funding, and travel accommodations from Celgene, as well as consultancy and travel accommodations from Gilead and Pharmacyclics and research funding from TG Therapeutics. S.J. receives consultancy and research funding from Pharmacyclics, consultancy funding from Seattle Genetics, and research funding from Celldex and Immunomedics. F.C. is employed by Pharmacyclics and holds stock in AbbVie. A.R.R. receives honoraria from WebMD and research funding from Pharmacyclics. L.S. is employed by Pharmacyclics and holds stock in AbbVie. S.E.C. receives consultancy funding from Janssen, consultancy and research funding from Pharmacyclics, and research funding from AbbVie. D.B.M. receives consultancy, research funding, and travel accommodations from Pharmacyclics, as well as research funding from Kite Pharma, Roche, and Novartis and travel accommodations from Sanofi Oncology. C.E.R., J.C.B., and M.J.K. declare no competing financial interests.

Correspondence: David B. Miklos, 269 West Campus Dr, CCSR 2205, Stanford, CA 94305; e-mail: dmiklos{at}stanford.edu.

Acknowledgments

The authors thank the patients who participated in this study, and their families, as well as all the Bone Marrow Transplant nurses, patient coordinators, and staff. The Pharmacyclics clinical trial data analysis was sponsored by Pharmacyclics, LLC, an AbbVie Company. Supriya Srinivasan provided editorial assistance, for which funding was provided by Pharmacyclics, LLC.

The Stanford work was supported by National Institutes of Health National Cancer Institute grant P01 CA049605 and Stanford University Cancer Institute–supported grant 1P030CA124435-01. D.B.M. and B.S. received research support from Pharmacyclics, LLC. C.E.R. received research support from the Stanford Medical Scholars Fellowship Program.

Footnotes

  • * C.E.R. and B.S. contributed equally to this study.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

  • Submitted June 20, 2016.
  • Accepted October 15, 2016.

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