Mepolizumab as a corticosteroid-sparing agent in lymphocytic variant hypereosinophilic syndrome

Article Outline

• Abstract
• Methods
  • Patients and blood samples
  • MHE100185 study design
  • Analysis of T-cell surface phenotype
  • Measurement of serum TARC and IL-5 levels
  • Assessment for T-cell clonality
  • Diagnosis of lymphocytic variant HES
  • Statistical analysis
• Results
  • Identification and characterization of patients with L-HES enrolled in the MHE100185 clinical trial
  • Mepolizumab trial end-point achievement in patients with L-HES
  • Identification of patients with increased serum TARC in the MHE100185 trial
  • Treatment response in patients with high versus normal serum TARC levels
• Discussion
• Acknowledgment
• Fig E1. 
• Table E1. 
• Table E2. 
• References
• Copyright

Background

Mepolizumab, a monoclonal anti–IL-5 antibody, is an effective corticosteroid-sparing agent for patients with Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)–negative hypereosinophilic syndrome (HES). Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T cells.

Objective

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 patients with HES and a normal T-cell profile.

Methods

Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and T-cell receptor gene rearrangement patterns, and their serum thymus-and-activation-regulated chemokine (TARC) levels were measured. Response to treatment was compared in patient subgroups based on results of these analyses.

Results

Lymphocytic variant HES was diagnosed in 13 of 63 patients with HES 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 patients with L-HES 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.

Conclusion

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 eosinophilopoietic factors.

Key words: CD3^-CD4⁺, lymphocytic variant HES, clonal T-cell, TARC, IL-5, mepolizumab

Abbreviations used: AITL, Angioimmunoblastic T-cell lymphoma, APC, Allophycocyanin, FITC, Fluorescein isothiocyanate, F/P, FIP1-like 1/platelet-derived growth factor receptor α fusion, HES, Hypereosinophilic syndrome, L-HES, Lymphocytic variant hypereosinophilic syndrome, PE, Phycoerythrin, PerCP, Peridinin chlorophyll protein complex, TARC, Thymus-and-activation-regulated chemokine, TCR, T-cell receptor, TRG, T-cell receptor γ

Hypereosinophilic syndromes (HESs) are rare disorders characterized by sustained, moderate-to-severe hypereosinophilia associated with eosinophil-induced tissue/organ damage and dysfunction.¹ 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 overproduction of IL-5 by type 2 T cells, leading to reactive polyclonal eosinophil expansion and activation.⁴

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 (ie, 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 thymus-and-activation-regulated chemokine (TARC) have also been described in patients with HES and evidence of IL-5 overproduction.⁹ Current treatment of L-HES relies mainly on corticosteroids, eventually combined with IFN-α; 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 corticosteroid-sparing agent in patients with corticosteroid-responsive, Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)–negative HES was recently demonstrated in the setting of a double-blind placebo-controlled clinical trial.¹¹ A significantly higher proportion of patients receiving mepolizumab were able to reduce their daily prednisone dose, and even to taper off corticosteroids completely, compared with those in the placebo arm. In addition, blood eosinophil levels were more tightly controlled in the active treatment arm, despite lower prednisone 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.

Because L-HES is characterized by increased T-cell production of IL-5 and patients with L-HES generally respond to corticosteroids, mepolizumab is a tempting alternative to conventional treatment. It is conceivable, however, that a higher dose of mepolizumab would be needed to neutralize the high levels of IL-5 effectively in patients with L-HES. To address this concern, we identified patients with L-HES in the mepolizumab trial and determined whether they showed corticosteroid 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.

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Methods 

Patients and blood samples 

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 corticosteroid-sparing agent in F/P-negative HES (clinical trial MHE100185; Clinicaltrials.gov identifier: NCT00086658). The trial and associated exploratory biomarker study were approved by each participating center's institutional review board. The clinical characteristics of the cohort are detailed in the original article reporting the results of the trial.¹¹ 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 (PreAnalytiX, Qiagen/BD, Hombrechtikon) 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 (Institute for Medical Immunology) for European samples for processing within 24 hours. Serum, PaxGene, and EDTA tubes were available for 81, 79, and 63 patients, respectively.

MHE100185 study design 

The design of the MHE100185 clinical trial has been described in detail elsewhere.¹¹ Briefly, patients were eligible for enrollment if they fulfilled Chusid’s criteria for HES, if they were F/P-negative, and if disease was controlled (eosinophil levels and clinical manifestations) by corticosteroid monotherapy at a daily prednisone dose between 20 and 60 mg. After randomization, patients received either intravenous mepolizumab 750 mg or placebo every 4 weeks. At week 1, prednisone was progressively tapered according to a predefined algorithm based on blood eosinophil levels and symptoms. The primary endpoint of the study was the ability to taper the daily prednisone dose to 10 mg or less for a period of at least 8 weeks. Secondary, post hoc, and exploratory endpoints addressed more profound and durable prednisone tapering as well as eosinophil control.

Analysis of T-cell surface phenotype 

Lymphocyte phenotyping was performed on whole blood within 24 hours by flow cytometry by using BD FacsCalibur and BD FACScan; (both 4-color) machines in Brussels and Utah, respectively, and CellQuest software (Becton Dickinson, NJ). The following fluorochrome-coupled antibodies were purchased from Becton Dickinson (same batches for both sites): Quadritest A (CD3–fluorescein isothiocyanate [FITC] CD8-phycoerythrin [PE ] CD45-peridinin chlorophyll protein complex [PerCP] CD4-allophycocyanin [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.

Measurement of serum TARC and IL-5 levels 

Frozen serum samples were sent to Brussels for serum TARC measurement and to Utah for measurement of IL-5. TARC was measured using the R&D Systems (Minn) TARC ELISA kit, and T_H2 cytokines were measured at Associated Regional and University Pathologists (ARUP) Laboratories (Salt Lake City, Utah); measurements were made in duplicate.

Assessment for T-cell clonality 

Frozen PaxGene tubes were sent to Bethesda, where TCR-γ (TRG) gene rearrangement studies were performed as described by Greiner and Rubocki.¹² PCR was performed by using primers that interrogate TRG rearrangements involving all of the known family members for variable segments of the gamma locus, 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 were analyzed by using GeneMapper software version 3.7 (ABI). This method can detect a clonal population made up of 2% to 5% of total T cells and identifies approximately 95% of all TRG rearrangements occurring in clonal T-cell proliferations.

Diagnosis of lymphocytic variant HES 

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 T_H2 cytokines at the cellular level (ie, CD3^-CD4⁺), or (2) flow cytometry showing an expanded population of unconventional T cells (eg, CD4⁺CD7^-) previously shown to produce T_H2 cytokines together with demonstrated clonality or oligoclonality.

Statistical analysis 

Nonparametric comparisons of group means and medians were made by using the Mann-Whitney U test. Proportions were compared by using the Fisher exact test. A P value below .05 was considered significant.

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Results 

Identification and characterization of patients with L-HES enrolled in the MHE100185 clinical trial 

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 I; Fig 1, A). 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 (see this article's Table E1 andFig E1 in the Online Repository at www.jacionline.org). 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 of 9 cases and a restricted (oligoclonal) pattern in 1 patient. Three additional patients had an expanded population of CD3⁺CD4⁺CD7^- T cells (patients 10-12, Table I; Fig 1, B), 1 with a clonal TCR rearrangement pattern and 2 with restricted patterns. A distinct CD3⁺CD8⁺CD5^lo subset was detected in 1 patient (patient 13, Table I; Fig 1, C) with a clonal TCR gene rearrangement pattern. Overall, criteria for L-HES were fulfilled in 13 of 63 (20.6%) patients for whom phenotyping studies were available (patients 1-13, Table I). None of these patients had lymphocytosis at baseline or during the study (at baseline, geometric mean, 1886/μL; range, 660-4240/μL). Clinically, skin involvement was the most frequent disease complication, especially in patients with CD3^-CD4⁺ cells (see this article's Table E2 in the Online Repository at www.jacionline.org). Other manifestations observed in more than half of cases involved the gastrointestinal tract, lungs, and muscles.

Table I. Patients enrolled in the MHE100185 study with an abnormal T-cell profile

F, Female; M, male; ND, not done.

This table includes all patients in whom T-cell phenotyping and/or PCR for TCR rearrangements showed abnormal results.

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• Fig 1. 

  Phenotypically aberrant T-cell subsets detected by flow cytometry on whole blood from patients enrolled in the MHE100185 trial. Phenotypically aberrant T-cell populations are shown within a closed shape (circle, box) on dot plots or are delimited by a horizontal bar (M1) on histograms. A, CD3^-CD4⁺ cells (plot gated on total lymphocytes according to forward and side scatter). B, CD3^-CD4⁺CD7^- cells (histogram gated on CD3⁺CD4⁺ lymphocytes). C, CD3⁺CD4^-(CD8⁺)CD5^lo/- (plot gated on CD3⁺CD4^- lymphocytes, which are mainly CD8⁺). D, CD3^-CD4⁺CD8^dim (plot gated on CD3⁺ lymphocytes). E, CD3^-CD4^dimCD8⁺ (plot gated on CD3⁺ lymphocytes). F, CD3⁺CD4⁺CD25^hi (histogram gated on CD3⁺CD4⁺ lymphocytes).

In addition to these well defined and previously reported phenotypes, expanded unconventional T-cell populations were observed in 2 additional cases (CD3⁺CD4⁺CD8^dim and CD3⁺CD4^dim CD8⁺, with clonal and restricted TCR rearrangement patterns, respectively; patients 14 and 15, Table I; Fig 1, D and E). In the absence of published evidence that double-positive T cells play a pathogenic role in HES through production of an eosinophilopoietic factor, these patients were classified as having idiopathic HES. An extremely elevated proportion of CD3⁺CD4⁺CD25^hi T cells was observed in 1 patient (patient 16, Table I; Fig 1, F), who was subsequently found to have mycosis fungoides.

T-cell receptor gene rearrangement studies were available for 79 patients. In addition to the patients with phenotypically aberrant T-cell lineage subsets described, a clonal TCR gene rearrangement pattern was revealed by PCR analysis in 3 patients (patients 17-19, Table I). We were unable to classify patient 17 because T-cell phenotyping was not performed, whereas patient 19 was classified as having 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).

Mepolizumab trial end-point achievement in patients with L-HES 

Of the 13 patients with an established L-HES diagnosis, 7 received mepolizumab and 6 received placebo. Subjects with L-HES receiving mepolizumab were significantly more likely to maintain a daily prednisone dose ≤10 mg for at least 24 weeks than the patients with L-HES receiving placebo (Fig 2) and had a lower mean daily prednisone dose at the end of the study (4.64 mg in the mepolizumab-treated group vs 28.23 mg in the placebo-treated group; P = .014). Four of 7 patients in the mepolizumab arm were corticosteroid-free at the end of the trial (vs 0/6 in the placebo group; P = .07). These findings were similar to those for the mepolizumab-treated study subjects without L-HES (Table II; comparisons were made only between patients with available T-cell phenotyping studies, excluding those with lymphoma). Subjects with L-HES 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% vs 100%, respectively; P = .045).

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• Fig 2. 

  Superiority of mepolizumab over placebo in patients with L-HES. The response rate of patients with L-HES to mepolizumab versus placebo is shown; white bars represent the mepolizumab treatment arm, and hatched bars represent the placebo treatment arm. PDN, Prednisone; wk, week; Eos, blood eosinophil level. ^∗P < .05; ^∗∗P < .005. For PDN 0 mg until end of study, 2-sided P value is .07 and 1-sided P value is .049 using the Fisher exact test.

Table II. Treatment responses in patients with L-HES versus a normal∗ T-cell profile

PDN, Prednisone; wk, week; PEP, primary endpoint; Eos, blood eosinophil level.

Two subjects who received mepolizumab (1 with L-HES and 1 without) failed to achieve the primary endpoint. The first patient, with a population of CD3^-CD4⁺ cells (patient 5, Table I), was unable to taper prednisone because of increasing eosinophilia associated with recurrence of symptoms, including angioedema and fever. He was considered a nonresponder and was withdrawn from the study. In contrast, the second subject, with a normal T-cell profile, was initially unable to taper prednisone despite normalization of peripheral eosinophilia, because of sinusitis and seasonal allergies. Prednisone taper was ultimately achieved, and the patient has been prednisone-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 endpoints.

Identification of patients with increased serum TARC in the MHE100185 trial 

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 having high TARC if baseline and/or peak serum TARC was above 1000 pg/mL. Increased TARC levels were recorded on at least 1 occasion in 33 of 81 (41%) subjects (Table III). Of the 33 patients with elevated TARC, 13 had L-HES (patients T1-T13, Table III), 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 having idiopathic HES; 7 had markedly increased serum IgE levels (>1000 kU/L), and 3 others had significantly elevated serum IL-5 levels.

Table III. Patients with increased serum TARC values

Patient	BL TARC∗	Peak TARC∗	Flow cytometry	TCR rearr	Serum IgE†	Serum IL-5‡	Diagnosis§
Patients with abnormal T-cell phenotype subset and/or T-cell clonality
T1	1,948	67,170	CD3-CD4+	Restricted	195	<7.8 (122.9)	L-HES (P1)
T2	31,312	31,312	CD3-CD4+	Clonal	9,195	43.4	L-HES (P2)
T3	15,788	15,788	CD3-CD4+	Clonal	121	10.6	L-HES (P3)
T4	74,895	98,511	CD3-CD4+	Clonal	7,215	<7.8 (8.4)	L-HES (P4)
T5	1,038	1,488	CD3-CD4+	Clonal	23	<7.8	L-HES (P5)
T6	7,020	35,463	CD3-CD4+	Clonal	255	178.5	L-HES (P6)
T7	ND	6,720	CD3-CD4+	Polyclonal	47	<7.8	L-HES (P7)
T8	5,559	13,924	CD3-CD4+	Clonal	1,083	139.6	L-HES (P8)
T9	137	20,705	CD3-CD4+	Clonal	4,032	<7.8	L-HES (P9)
T10	858	7,529	CD4+CD7-	Restricted	2,149	<7.8	L-HES (P10)
T11	849	1,488	CD4+CD7-	Restricted	150	<7.8	L-HES (P11)
T12	416	2,830	CD4+CD7-	Clonal	1,445	<7.8 (32.8)	L-HES (P12)
T13	390	1,430	CD3+CD8+CD5lo/neg	Clonal	424	<7.8	L-HES (P13)
T14	6,200	19,090	CD3+CD4+CD25hi	Polyclonal	12	<7.8	CTCL (P16)
T15	32,618	218,500	ND	Clonal	71,540	<7.8	AITL (P18)
T16	583	2,543	CD3+CD4+CD8dim	Clonal	158	<7.8	I-HES (P14)
T17	1,862	128,743	ND	Clonal	124	<7.8	I-HES (P17)
Patients with no overt T-cell abnormalities
T18	1,035	1,035	ND	Polyclonal	101	<7.8	I-HES
T19	1,049	1,770	Nl	Polyclonal	1,115	<7.8	I-HES
T20	229	1,741	Nl	Polyclonal	170	<7.8 (78)	I-HES
T21	2,311	2,311	CD4/CD8: 6	Polyclonal	44	177.4	I-HES
T22	169	1,930	CD4/CD8: 6.8	Polyclonal	29	<7.8 (302.5)	I-HES
T23	726	1,009	Nl	Polyclonal	302	<7.8	I-HES
T24	481	1,398	CD4/CD8 <1	Clonal	34	<7.8	I-HES
T25	557	6,790	CD4/CD8: 8	Polyclonal	2,495	<7.8	I-HES
T26	ND	1,023	Nl	Polyclonal	214	15	I-HES
T27‖	206	10,624	ND	Polyclonal	2,160	11.6	I-HES
T28	2,040	6,470	CD4/CD8: 5.9	Restricted	116	<7.8 (9.9)	I-HES
T29	1,619	35,413	Nl	Polyclonal	3,122	<7.8	I-HES
T30	818	1,492	Nl	Polyclonal	49	23	I-HES
T31	ND	2,721	Nl	Polyclonal	1,700	<7.8	I-HES
T32	7,330	9,130	ND	Polyclonal	10,740	<7.8	I-HES
T33	303	1,208	ND	Polyclonal	17,185	<7.8 (15.2)	I-HES

AITL, Angioimmunoblastic T cell lymphoma; BL, baseline; CTCL, cutaneous T-cell lymphoma; I-HES, idiopathic HES; ND, not done; Nl, normal; rearr, rearrangement.

This table includes all patients in whom peak serum TARC was above 1000 pg/mL.

Treatment response in patients with high versus normal serum TARC levels 

Response to treatment was compared between high TARC patients versus those with TARC levels below 1000 pg/mL (Table IV). In the mepolizumab treatment arm, only the ability to maintain eosinophils below 600/μL throughout the study was significantly different between the 2 groups (11/15 in the high TARC group vs 24/24 in the group with lower TARC levels; P = .017). In the placebo treatment arm, no significant differences were observed using this cutoff level for serum TARC.

Table IV. Treatment responses in patients with HES with peak serum TARC levels above versus below 1000 pg/mL

PDN, Prednisone; wk, week; PEP, primary endpoint; Eos, blood eosinophil level.

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Discussion 

The current study took advantage of widespread recruitment of patients with corticosteroid-responsive HES for participation in a clinical trial 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 on the basis of recent studies showing clonal TCR rearrangements in high proportions of patients with HES, 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 al⁷ (16/60; 25%) in a cohort of patients with HES 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%.¹⁵ The relatively high proportion of phenotypically abnormal T-cell subsets observed in our study can be explained by exclusion of patients with F/P-associated HES from the clinical trial and by selection of patients who responded to corticosteroid monotherapy, which likely enriched the cohort for patients with L-HES 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, clonal or oligoclonal TCR rearrangement patterns were observed in some patients with phenotype abnormalities that have not yet been investigated in the setting of HES (eg, 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-4,¹⁶ may be increased in various conditions associated with in vivo T_H2-cell activation, including L-HES.⁹, ¹⁷, ¹⁸, ¹⁹, ²⁰ We therefore measured serum TARC to identify additional patients with presumed T_H2-driven disease in this study, beyond those with clear-cut phenotype abnormalities. Forty percent (33/81) of patients with HES in this study had a peak serum TARC level above 1000 pg/mL (Table III), including all 13 patients with phenotypically proven L-HES although they were treated with corticosteroids, and even when the proportion of aberrant cells was very low (less than 2.5% lymphocytes in 3 cases).

Mepolizumab was an effective corticosteroid-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 prednisone endpoints (Table II). Remarkably, approximately half of the patients were able to taper completely off corticosteroids 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 (Table II, Table IV). 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 T_H2-mediated disease. Indeed, 2 of them had CD3^-CD4⁺ L-HES (patients 5 and 6, Table I), 1 had a clonal TCR rearrangement pattern and extremely elevated serum TARC (patient 17, Table I), and the fourth patient (patient T27, Table III) 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). For patients 6 and 17, the rise in eosinophilia was easily overcome with low-dose prednisone maintenance therapy (<10 mg daily); in these conditions, HES-related clinical manifestations were well controlled, and eosinophils never exceeded 1000/μL (Table E2). In contrast, patients 5 and T27 were truly resistant to mepolizumab, with uncontrolled eosinophilia and recurrent HES-related symptoms as prednisone tapering was attempted, resulting in permanent withdrawal from the study in 1 case and HES-related death in the other.

Mepolizumab's failure to deplete eosinophils in these cases likely reflects incomplete neutralization of IL-5 because of marked IL-5 overproduction in vivo and/or antibody-inaccessible local production of IL-5; and possibly uninhibited effects of other eosinophilopoietic 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, ⁵, ⁶, ⁷, ²¹ and PBMCs from patients with increased serum TARC,⁹ produce more IL-5 in vitro than CD4 T cells and PBMCs from control subjects and other patients with HES. Further evidence that increased TARC levels are indicative of a stronger eosinophilopoietic 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 corticosteroid-tapering period (90% vs 46%; P = .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 corticosteroid maintenance therapy.

Overall, these results indicate that mepolizumab (1) represents an effective corticosteroid-sparing agent for the majority of patients with T-cell–driven HES (ie, L-HES and/or increased serum TARC), similar to other patients with HES, 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,¹¹ mepolizumab represents a very tempting alternative to IFN-α, the currently preferred but poorly tolerated corticosteroid-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 during corticosteroid dose reduction for early detection of resistance or requirement of a prednisone maintenance dose. Maintenance of low-dose corticosteroid treatment may actually be preferable for all patients with L-HES under mepolizumab, because, in contrast with corticosteroids and IFN-α,¹⁰ 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 E1), and there is reason to believe that both mepolizumab itself²² and the corticosteroid tapering it enables may increase their production of T_H2 cytokines.

Finally, although the 2 patients with T-cell lymphoma were excluded from our analyses, we underscore that neither reached the primary endpoint under mepolizumab. The patient with mycosis fungoides presented persistent marked erythroderma requiring continued corticosteroid treatment despite effective depletion of blood and skin-infiltrating eosinophils with mepolizumab, whereas the patient with AITL responded in terms of neither 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 because of 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 antieosinophil strategies may unmask and even accelerate progression of such diseases by enabling corticosteroid 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, because these T cells may be premalignant and are sensitive to the proapoptotic effects of corticosteroids. 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 patients with HES with phenotypically abnormal T-cell subsets and/or increased serum TARC levels respond to mepolizumab and are able to taper corticosteroids at a rate that is comparable to that of the overall HES population. However, in contrast with 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 eosinophilopoietic factors are not fully neutralized. Furthermore, corticosteroid tapering on mepolizumab may unmask corticosteroid-suppressed T-cell disease. Pursuing low-dose corticosteroids may therefore be prudent in patients with L-HES to target the potentially premalignant T cells, in parallel with eosinophil-directed therapy.

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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 with GlaxoSmithKline, who played a key role in the logistical set-up of the exploratory studies; Ann Haig and Steve Mallett, currently with GlaxoSmithKline, for pursuing close collaboration and providing a wealth of data from the clinical trial; and Elaine Griffin with Envision for critical review of the article.

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Fig E1. 

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• Histograms represent expression of surface antigens by normal CD3⁺CD4⁺ T cells (hashed line) and abnormal CD3^-CD4⁺ T cells (filled or bold histograms) from patient 6 (Table I).

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Table E1. 

Detailed phenotype of CD3^-CD4⁺ T cells

ND, Not done, phenotyping was only available at baseline.

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Table E2. 

Clinical characteristics and treatment of patients with L-HES

Tx arm, Treatment arm; PDN, prednisone; BL, baseline; Max, maximum; End, end of study.

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