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Mepolizumab, a monoclonal anti-interleukin-5 (IL-5) antibody, is an effective corticosteroid-sparing agent for patients with F/P-negative hypereosinophilic syndrome. Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T-cells.
To determine whether patients with L-HES respond to mepolizumab in terms of corticosteroid tapering and eosinophil depletion to the same extent as corticosteroid-responsive F/P-negative HES patients with a normal T-cell profile.
Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and TCR gene rearrangement patterns, and their serum thymus-and-activation-regulated (TARC) chemokine levels were measured. Response to treatment was compared in patient subgroups, based on results of these analyses.
L-HES was diagnosed in 13 of 63 HES patients with complete T-cell assessments. The ability to taper corticosteroids on mepolizumab was similar in patients with L-HES and those with a normal T-cell profile, although a lower proportion of L-HES patients maintained eosinophil levels below 600/μl. Increased serum TARC levels (>1000 pg/ml) had no significant impact on the ability to reduce corticosteroid doses, but a lower proportion of patients with elevated TARC achieved eosinophil control on mepolizumab.
Mepolizumab is an effective corticosteroid-sparing agent for patients with L-HES. In some cases however, eosinophil levels remain above 600/μl, suggesting incomplete neutralization of overproduced IL-5 or involvement of other eosinophilopoïetic factors.
Mepolizumab is an effective corticosteroid-sparing agent for the majority of patients with L-HES and/or increased serum TARC, but fails to maintain eosinophils below 600/μl in a subset of such patients.
Hypereosinophilic syndromes (HES) are rare disorders, characterized by sustained, moderate-to-severe hypereosinophilia associated with eosinophil-induced tissue/organ damage and dysfunction . The term encompasses very heterogeneous clinical presentations with respect to the nature and severity of eosinophil-mediated complications, overall prognosis, and response to therapy [2, 3]. Distinct pathogenic mechanisms have now been identified, allowing for classification of some patients with HES into more homogeneous and “predictable” disease subsets. One of these entities, termed “lymphocytic variant” HES (L-HES), is characterized by over-production of interleukin (IL)-5 by “type 2” T-cells, leading to “reactive” polyclonal eosinophil expansion and activation .
Clinically, patients with L-HES often have predominantly cutaneous manifestations, and long-term follow-up has shown malignant transformation of T-cells in several cases. Diagnosis of L-HES currently relies on combined analysis of T-cell phenotypes and T-cell receptor (TCR) gene rearrangement patterns. Early case studies indeed identified monoclonal T-cell subsets with abnormal expression of surface markers (i.e. TCRα/β-CD3-CD4+ and CD3+CD4-CD8-) that produced high levels of IL-5 in vitro, suggesting their involvement in hypereosinophilia [5, 6]. Subsequently, other unusual T-cell phenotypes have been reported in association with HES [4, 7, 8]. Elevated serum levels of TARC have also been described in patients with HES and evidence of IL-5 over-production . Current treatment of L-HES relies mainly on corticosteroids (CS), eventually combined with IFNα; both agents target eosinophils and cytokine production by the abnormal T-cells [4, 10].
Mepolizumab is a monoclonal anti-IL-5 antibody, which binds to free IL-5 and interferes with engagement of the IL-5 receptor on eosinophils. The efficacy of mepolizumab as a CS-sparing agent in patients with CS-responsive, FIP1L1/PDGFRA (F/P)-negative HES was recently demonstrated in the setting of a double-blind placebo-controlled clinical trial . A significantly higher proportion of patients receiving mepolizumab were able to reduce their daily prednisone (PDN) dose, and even to taper off CS completely, compared with those in the placebo arm. In addition, blood eosinophil levels were more tightly controlled in the active treatment arm, despite lower PDN doses, suggesting that mepolizumab efficiently targets eosinophil expansion in patients with HES. Importantly, mepolizumab was well-tolerated with no significant differences in treatment-related adverse events relative to placebo.
Since L-HES is characterized by increased T-cell production of IL-5 and patients with L-HES generally respond to CS, mepolizumab is a tempting alternative to conventional treatment. It is conceivable, however, that a higher dose of mepolizumab would be needed to effectively neutralize the high levels of IL-5 in patients with L-HES. To address this concern, we identified patients with L-HES in the mepolizumab trial, and determined whether they showed CS tapering and eosinophil depletion to the same extent as the patients with a normal T-cell profile. We also investigated whether increased serum TARC concentrations, a possible surrogate marker for the presence of activated type 2 cytokine-producing cells, predicted response to therapy, irrespective of the presence of a clearly defined abnormal T-cell population.
Eighty-five patients with HES were recruited for inclusion in an international, randomized, double-blind, placebo-controlled clinical trial to investigate the efficacy of mepolizumab as a CS-sparing agent in F/P-negative HES (clinical trial MHE100185; Clinicaltrials.gov identifier: NCT00086658). The trial and associated “exploratory biomarker” study was approved by each participating center’s IRB. The clinical characteristics of the cohort are detailed in the original paper reporting the results of the trial . Patients marked their agreement to this biomarker study by ticking a specific box in the consent form. Two sets of blood samples were drawn at baseline, and at weeks 12, 24 and 36 (or at withdrawal) for the biomarker study. The first, serum and PaxGene tubes for measurement of soluble factors in serum and for PCR analysis of T-cell clonality, was sent to a central laboratory for storage at -20°C. The second, an EDTA tube for T-cell phenotyping by flow cytometry, was sent separately to investigators at University of Utah for North American samples and Université Libre de Bruxelles for European samples for processing within 24 hours. Serum, PaxGene, and EDTA tubes were available for 81, 79, and 63 patients, respectively.
The design of the MHE100185 clinical trial has been described in detail elsewhere . Briefly, patients were eligible for enrollment if they fulfilled Chusid’s criteria for HES, were F/P-negative, and if disease was controlled (eosinophil levels and clinical manifestations) by CS-monotherapy, at a daily PDN dose between 20 and 60 mg. After randomization, patients received either intravenous mepolizumab 750 mg or placebo every 4 weeks. At week 1, PDN was progressively tapered according to a pre-defined algorithm, based on blood eosinophil levels and symptoms. The primary end-point of the study was the ability to taper the daily PDN dose to 10 mg or less, for a period of at least 8 weeks. Secondary, post-hoc and exploratory end-points addressed more profound and durable PDN tapering, as well as eosinophil control.
Lymphocyte phenotyping was performed on whole blood within 24 hours by flow cytometry, using FacsCalibur (Brussels) and BD FACScan™ (Utah) (4-color) machines and CellQuest software. The following fluorochrome-coupled antibodies were purchased from Becton Dickinson (same batches for both sites): Quadritest A (CD3-FITC CD8-PE CD45-PerCP CD4-APC), Quadritest B (CD3-FITC CD16/56-PE CD45-PerCP CD19-APC), CD3-PerCP, CD4-APC, TCRαβ-FITC, CD5-PE, CD7-FITC, CD25-PE, CD2-FITC, and HLA-DR-PE. Appropriate control isotypes were used. Staining and acquisition were performed in Brussels and in Utah, and when the study was completed, acquisition files were centralized in Belgium for analysis.
Frozen serum samples were sent to Brussels for serum TARC measurement, and to Utah for measurement of IL-4 and IL-5. TARC was measured using the R&D TARC ELISA kit, and Th2 cytokines were measured at ARUP Laboratories (Salt Lake City, Utah); measurements were made in duplicate.
Frozen PaxGene tubes were sent to Bethesda, where TCRgamma (TRG) gene rearrangement studies were performed as described by Greiner et al . PCR was performed using primers that interrogate TRG rearrangements involving all of the known Vg family members, and the Jg1/2, JP1/2 and JP joining segments. To allow for fluorescence detection, each joining region primer was covalently linked to a unique fluorescent dye. The products were analyzed by capillary electrophoresis on an ABI 3130xl Genetic Analyzer, and electropherograms analyzed using GeneMapper software version 3.7 (ABI). This method can detect a clonal population comprising 2-5% of total T-cells, and identifies approximately 95% of all TRG rearrangements occurring in clonal T-cell proliferations.
Diagnosis of L-HES was based on results of T-cell phenotyping and TCR gene rearrangement patterns. The following conditions had to be met: 1) flow cytometry showing a well-circumscribed, phenotypically aberrant subset previously shown to produce Th2 cytokines at cellular level (i.e. CD3-CD4+), or 2) flow cytometry showing an expanded population of unconventional T-cells (e.g. CD4+CD7-) previously shown to produce Th2 cytokines together with demonstrated (oligo)clonality.
Non-parametric comparisons of group means and medians were made using the Mann-Whitney U test. Proportions were compared using Fisher’s exact test. A p-value below 0.05 was considered significant.
Analysis of T-cell surface antigens by flow cytometry on peripheral blood from 63 patients enrolled in the mepolizumab trial revealed that 9 patients (14%) had a well-circumscribed CD3-CD4+ T-cell lineage subset (patients 1-9, Table 1; Figure 1a). As reported in previous studies, the CD3-CD4+ cells showed negative staining for TCRα/β, intense staining for CD5 compared with the normal CD3+CD4+ cells in the same sample, and lower overall CD7 expression (supplementary Table S1 and Figure S1). Absolute numbers of CD3-CD4+ cells at baseline ranged from 15 to 2914 cells/μl. Assessment of T-cell clonality by PCR analysis of TCR Vγ chains showed clonal rearrangement patterns in 7/9 cases and a restricted (oligoclonal) pattern in one patient. Three additional patients had an expanded population of CD3+CD4+CD7- T-cells (patients 10-12, Table 1; Figure 1b), one with a clonal TCR rearrangement pattern and two with restricted patterns. A distinct CD3+CD8+CD5lo subset was detected in one patient (patient 13, Table 1; Figure 1c) with a clonal TCR gene rearrangement pattern. Overall, criteria for L-HES were fulfilled in 13/63 (20.6%) patients for whom phenotyping studies were available (P1-P13, Table 1). None of these patients had lymphocytosis at baseline or during the study (at baseline, GM 1886/μl, range 660-4240/μl). Clinically, skin involvement was the most frequent disease complication, especially in patients with CD3-CD4+ cells (Table S2). Other manifestations observed in more than half of cases involved the gastrointestinal tract, lungs, and muscles.
In addition to these well-defined and previously reported phenotypes, expanded unconventional T-cell populations were observed in 2 additional cases (CD3+CD4+CD8dim and CD3+CD4dim CD8+, with clonal and restricted TCR rearrangement patterns, respectively; patients 14-15, Table 1; Figure 1d-e). In the absence of published evidence that double positive T-cells play a pathogenic role in HES through production of an eosinophilopoïetic factor, these patients were classified as idiopathic HES. An extremely elevated proportion of CD3+CD4+CD25hi T-cells was observed in one patient (patient 16, Table 1; Figure 1f), who was subsequently found to have mycosis fungoïdes.
TCR gene rearrangement studies were available for 79 patients. In addition to the patients with phenotypically aberrant T-cell lineage subsets described above, a clonal TCR gene rearrangement pattern was revealed by PCR analysis in 3 patients (patients 17-19, Table 1). We were unable to classify patient 17 because T-cell phenotyping was not performed, whereas patient 19 was classified as idiopathic HES on the basis of unremarkable phenotyping studies. Patient 18, who had extremely elevated serum TARC (218,500 pg/ml), was ultimately diagnosed with angioimmunoblastic T-cell lymphoma (AITL).
Of the 13 patients with an established L-HES diagnosis, 7 received mepolizumab and 6 received placebo. L-HES subjects receiving mepolizumab were significantly more likely to maintain a daily PDN dose ≤10 mg for at least 24 weeks than the L-HES patients receiving placebo (Figure 2), and had a lower mean daily PDN dose at the end of the study (4.64 mg in the mepolizumab-treated group, versus 28.23 mg in the placebo-treated group [p=0.014]). Four of 7 patients in the mepolizumab arm were CS-free at the end of the trial (versus 0/6 in the placebo group, p=0.07). These findings were similar to those for the mepolizumab-treated study subjects without L-HES (Table 2; comparisons were made only between patients with available T-cell phenotyping studies, excluding those with lymphoma). L-HES subjects treated with mepolizumab were also more likely than those receiving placebo to achieve an eosinophil count below 600/μl for 8 weeks and for the duration of the trial, but were less likely to maintain eosinophil levels below 600/μl during the entire trial than mepolizumab-treated subjects without L-HES (71% versus 100%, respectively; p=0.045).
Two subjects who received mepolizumab (one with L-HES and one without) failed to achieve the primary end-point. The first patient, with a population of CD3-CD4+ cells (P5, Table 1), was unable to taper PDN because of increasing eosinophilia associated with recurrence of symptoms, including angioedema and fever. He was considered a non-responder and was withdrawn from the study. In contrast, the second subject, with a normal T-cell profile, was initially unable to taper PDN despite normalization of peripheral eosinophilia, because of sinusitis and seasonal allergies. PDN taper was ultimately achieved and he (she) has been PDN-free for the past 3 years in the open-label extension study (not shown).
In the placebo treatment arm, no significant differences were observed between subjects with and without L-HES with respect to any of the trial end-points.
Serum TARC levels were measured at baseline and at 12-week intervals in 81 subjects. Baseline, as well as “peak,” serum TARC levels were recorded for each patient. Patients were classified as “high TARC” if baseline and/or peak serum TARC was above 1000 pg/ml. Increased TARC levels were recorded on at least one occasion in 33/81 (41%) subjects (Table 3). Of the 33 patients with elevated TARC, 13 had L-HES (patients T1-T13, Table 3), 2 were retrospectively diagnosed with T-cell lymphoma after inclusion in the trial, and 2 had a clonal TCR rearrangement pattern, but did not fulfill criteria for L-HES because phenotyping was either unremarkable or unavailable. The remaining 16 patients were classified as idiopathic HES; 7 had markedly increased serum IgE levels (>1000 KU/L), and 3 others had significantly elevated serum IL-5 levels.
Response to treatment was compared between “high TARC” patients versus those with TARC levels below 1000 pg/ml (Table 4). In the mepolizumab treatment arm, only the ability to maintain eosinophils below 600/μl throughout the study was significantly different between the two groups (11/15 in the “high TARC” group, versus 24/24 in the group with lower TARC levels; p=0.017). In the placebo treatment arm, no significant differences were observed using this cut-off level for serum TARC.
The current study took advantage of widespread recruitment of patients with steroid-responsive HES for participation in a clinical trial, in order to estimate the proportion of patients with L-HES and/or increased serum TARC values in a large cohort, and to explore responses of patients with T-cell driven disease to highly targeted eosinophil-specific therapy. Diagnosis of L-HES was based on detection of abnormal T-cell phenotypes associated with evidence of clonal T-cell expansion. Demonstration of T-cell clonality alone was not considered sufficient evidence for L-HES, based on recent studies showing clonal TCR rearrangements in high proportions of HES patients, the majority of whom have normal T-cell phenotypes and no evidence of increased IL-5 production [13, 14]. The proportion of patients with L-HES included in this clinical trial (13/63) was approximately 20%, which is similar to that reported by Simon et al (16/60 [25%]) in a cohort of HES patients referred mainly to dermatologists . A recent, large retrospective multicenter study, designed to minimize the referral bias of single-center studies, reported 17% patients with L-HES, but the actual proportion with well-documented T-cell phenotype abnormalities was only 8.7% . The relatively high proportion of phenotypically abnormal T-cell subsets observed in our study can be explained by exclusion of patients with FIP1L1/PDGFRA-associated HES from the clinical trial, and by selection of patients who responded to CS-monotherapy, which likely enriched the cohort for L-HES patients compared with more aggressive, presumably myeloproliferative, disease forms.
Results of T-cell analyses on this patient cohort illustrate the heterogeneity within L-HES and how challenging formal diagnosis of this variant has become. In addition to patients with previously well-characterized phenotypically aberrant T-cell subsets, (oligo)clonal TCR rearrangement patterns were observed in some patients with phenotype abnormalities which have not yet been investigated in the setting of HES (e.g. double positive T-cells). Although demonstration of increased IL-5 production at the single-cell level should clarify the possible pathogenic role of these T-cell subsets, this is not within the bounds of routine explorations, and biomarkers of T-cell driven hypereosinophilia, which override phenotypic and functional differences, are desperately needed. Previous studies show that TARC, which is produced by various cell-types in response to IL-4 , may be increased in various conditions associated with in vivo Th2-cell activation, including L-HES [9, 18-21]. We therefore measured serum TARC to identify additional patients with presumed Th2-driven disease in this study, beyond those with clear-cut phenotype abnormalities. Forty percent (33/81) of HES patients in this study had a peak serum TARC level above 1000 pg/ml (Table 3), including all 13 patients with phenotypically proven L-HES although they were under CS, and even when the proportion of aberrant cells was very low (less than 2.5% lymphocytes in 3 cases).
Mepolizumab was an effective CS-sparing agent in patients with L-HES and/or increased serum TARC, with response rates similar to patients with a normal T-cell profile for the primary and more stringent PDN end-points (Table 2). Remarkably, approximately half of the patients were able to taper completely off CS by the end of the study, regardless of their T-cell profile. In contrast, a lower proportion of patients with L-HES and/or serum TARC above 1000 pg/ml durably maintained eosinophil levels below 600/μl during mepolizumab therapy (Tables 2 and and4).4). Viewed differently, the only 4 patients in the trial whose eosinophil levels increased above 600/μl despite mepolizumab had increased serum TARC and clear-cut evidence for Th2-mediated disease. Indeed, 2 of them had CD3-CD4+ L-HES (patients 5 and 6, Table 1), 1 had a clonal TCR rearrangement pattern and extremely elevated serum TARC (patient 17, Table 1), and the fourth patient had increased serum TARC and IgE levels, as well as the highest serum IL-4 level observed in this study (125 pg/ml, not shown) (patient T27, Table 3). For patients 6 and 17, the rise in eosinophilia was easily overcome with low-dose PDN maintenance therapy (<10 mg daily); in these conditions, HES-related clinical manifestations were well-controlled, and eosinophils never exceeded 1000/μl (Table S2). In contrast, patients 5 and T27 were truly resistant to mepolizumab, with uncontrolled eosinophilia and recurrent HES-related symptoms as PDN-tapering was attempted, resulting in permanent withdrawal from the study in one case, and HES-related death in the other. Mepolizumab’s failure to deplete eosinophils in these cases likely reflects incomplete neutralization of IL-5, due to marked IL-5 over-production in vivo and/or antibody-inaccessible local production of IL-5; and possibly uninhibited effects of other eosinophilopoïetic factors produced by dysregulated T-cells. Although T-cell production of IL-5 was not assessed in the current study, several publications demonstrate that CD3-CD4+, CD3+CD4+CD7-, and CD3+CD8+CD5- T-cells from patients with L-HES [4-7, 16], and PBMC from patients with increased serum TARC , produce more IL-5 in vitro than CD4 T-cells and PBMC from control subjects and other HES patients. Further evidence that increased TARC levels are indicative of a stronger eosinophilopoïetic drive is provided in the placebo-treatment arm, wherein a higher proportion of patients with serum TARC above 2000 pg/ml experienced recurrence of hypereosinophilia within 8 weeks during the initial CS-tapering period (90% vs 46%, p=0.025, data not shown). The ongoing open-label extension study should clarify the long-term practical implications of these findings, namely the impact on intervals between mepolizumab infusions, as well as the requirement for CS maintenance therapy.
Overall, these results indicate that mepolizumab 1) represents an effective CS-sparing agent for the majority of patients with T-cell driven HES (i.e. L-HES and/or increased serum TARC), similar to other HES patients, but 2) may provide only incomplete disease control as monotherapy in a subset of these patients, whose eosinophil levels remain above 600/μl. Given its excellent tolerance and safety profile , mepolizumab represents a very tempting alternative to IFNα, the currently preferred but poorly tolerated CS-sparing agent for this HES variant. This study indicates that once mepolizumab is initiated, blood eosinophil levels and clinical status should, nevertheless, be monitored closely as CS doses are reduced for early detection of resistance or requirement of a PDN maintenance dose. Maintenance of low-dose CS may actually be preferable for all patients with L-HES under mepolizumab, because, in contrast to CS and IFNα , mepolizumab is not expected to exert inhibitory effects on the pathogenic T-cells involved in L-HES. Indeed, the abnormal T-cells remain detectable in peripheral blood throughout the study (Table S1) and there is reason to believe that both mepolizumab itself  and the CS-tapering it enables may increase their production of Th2 cytokines.
Finally, although the 2 patients with T-cell lymphoma were excluded from our analyses, we underscore that neither reached the primary end-point under mepolizumab. The patient with mycosis fungoïdes presented persistent marked erythroderma requiring continued CS despite effective depletion of blood and skin-infiltrating eosinophils with mepolizumab, whereas the patient with AITL responded neither in terms of disease manifestations nor eosinophil levels. Both received appropriate therapy for their lymphoma; the first is in remission, and the second died a week later of cardiorespiratory failure, due to heart infiltration by the malignant T-cells. The fact that 2 patients with high serum TARC levels initially diagnosed with HES were ultimately diagnosed with lymphoma suggests that increased serum TARC should prompt careful assessment for underlying lymphoma, both at diagnosis and at regular intervals. Furthermore, it is conceivable that targeted anti-eosinophil strategies may unmask and even accelerate progression of such diseases by enabling CS tapering in some patients, and thereby alleviating some of the pressure on abnormal T-cells. This possibility is of utmost relevance for patients with L-HES, namely CD3-CD4+ associated disease, as these T-cells may be pre-malignant, and are sensitive to the pro-apoptotic effects of CS. Prolonged follow-up of patients with L-HES on mepolizumab therapy in the open-label extension study should provide some insight into the effects of treatment on numbers and activation status of pathogenic T-cell subsets.
In conclusion, this study shows that HES patients with phenotypically abnormal T cell subsets and/or increased serum TARC levels respond to mepolizumab and are able to taper CS at a rate that is comparable to that of the overall HES population. However, in contrast to patients with normal T-cell profiles, mepolizumab fails to deplete eosinophils in a subset of patients with T-cell driven disease, suggesting that overproduced IL-5 and/or other eosinophilopoïetic factors are not (fully) neutralized. Furthermore, CS-tapering on mepolizumab may unmask CS-suppressed T cell disease. Pursuing low-dose CS may therefore be prudent in L-HES patients, in order to target the potentially pre-malignant T-cells, in parallel with eosinophil-directed therapy.
We thank Martine Ducarme and Myriam Libin from Brussels, Saritha Kalva from Utah, and Thu Anh Pham from Maryland for expert technical assistance. We are also very grateful to all investigators involved in the mepolizumab clinical trial for complying with the increased complexity this exploratory study imposed. We thank Jeff Wilkins, formerly from GlaxoSmithKline, who played a key role in the logistical set-up of the exploratory studies; Ann Haig and Steve Mallett, currently GlaxoSmithKline, for pursuing close collaboration and providing a wealth of data from the clinical trial; and Elaine Griffin from Envision for critical reviewing of the manuscript.
Sources of funding: GlaxoSmithKline purchased the monoclonal antibodies for flow cytometry, and sponsored the MHE100185 clinical trial (Clinicaltrials.gov identifier: NCT00086658) through which patients were recruited for the present study. This study was supported by the Belgian National Fund for Scientific Research (FNRS) - through FRSM grant 3.4.582.05.4 and Télévie grant 7.4.573.08.F - and received funding from the David and Alice Van Buuren Foundation (Université Libre de Bruxelles). This work was funded, in part, by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases and National Cancer Institute, and work at the University of Utah was supported through National Institutes of Health grant AI-061097.
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