The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRα-positive hypereosinophilic syndrome. Results of a multicenter prospective study

Abstract

Background and Objectives The hypereosinophilic syndrome (HES) may be associated with the fusion of the platelet derived growth factor receptor α (PDGFRα) gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated PDGFRα tyrosine kinase. These cases with FIP1L1-PDGFRα rearrangement have been reported to be very sensitive to the tyrosine kinase inhibitor imatinib mesylate.Design and Methods A prospective multicenter study of idiopathic or primary HES was established in 2001 (Study Protocol Registration no. NCT 0027 6929). One hundred and ninety-six patients were screened, of whom 72 where identified as having idiopathic or primary HES and 63 were treated with imatinib 100 to 400 mg daily.Results Twenty-seven male patients carried the FIP1L1-PDGFRα rearrangement. All 27 achieved a complete hematologic remission (CHR) and became negative for the fusion transcripts according to reverse transcriptase polymerase chain reaction (RT-PCR) analysis. With a median follow-up of 25 months (15–60 months) all 27 patients remain in CHR and RT-PCR negative, and continue treatment at a dose of 100 to 400 mg daily. In three patients imatinib treatment was discontinued for few months, the fusion transcript became rapidly detectable, and then again undetectable upon treatment reassumption. Thirty-six patients did not carry the rearrangement; of these, five (14%) achieved a CHR, which was lost in all cases after 1 to 15 months.Interpretation and Conclusions All patients meeting the criteria for idiopathic or primary HES should be screened for the FIP1L1-PDGFRα rearrangement. For all patients with this rearrangement, chronic imatinib treatment at doses as low as 100 mg daily ensures complete and durable responses.

Thee term hypereosinophilic syndrome (HES) covers a relatively wide range of conditions in which eosinophilia is apparently primary or idiopathic (i.e. is not associated with other recognizable disease entities), is consistent (more than 1.5×10 eosinophils per liter), is durable (for more than 6 months), and may be associated with symptoms and signs of organ or tissue involvement and dysfunction.1 So far HES treatment, including corticosteroids, hydroxyurea, and interferon-α, has been symptomatic or palliative, and of limited success.1, 2 The causes and the nature of such cases of primary eosinophilia remained obscure until it was shown that in some cases of eosinophilia associated with a myelodysplastic syndrome the platelet derived growth factor receptor beta (PDGFRβ) was constitutively activated upon fusion with different partner genes, so providing a cytokine independent signal for cell proliferation.3–8 These rare cases of MDS are exquisitely responsive to the protein tyrosine kinase inhibitor imatinib mesylate, which is able to inhibit the kinase activity of PDGFR‚ at nanomolar concentrations.9, 10 Soon after, imatinib was reported to be effective also in the treatment of other patients with primary eosinophilia,11, 15 and it was rapidly found that in these patients an interstitial deletion in chromosome 4 led to the formation of a novel gene by fusion of the FIP1L1 gene with the PDGFRα gene, coding for a tyrosine kinase that is a constitutively activated form of PDGFRα,16–18 and can also be inhibited by imatinib at nanomolar concentrations.10 The cases of HES that are based on this peculiar molecular abnormality may be renamed as cases of chronic eosinophilic leukemia (CEL).2, 19 In a short time, several such cases were identified and were shown to be very sensitive to imatinib.17, 30, 31 There is fairly good evidence that imatinib is likely to be the treatment of choice of FIP1L1-PDGFRα rearranged HES/CEL, but the number of cases is small, and the long-term results of treatment are unknown. We report here on 27 patients with the FIP1L1-PDGFRα rearrangement who have been treated with imatinib for 15 to 60 months (median 25 months).

Design and Methods

Study and treatment protocol

A prospective study and treatment protocol of primary eosinophilia was approved by the Ethics Committee of the S. Orsola-Malpighi University Hospital in 2001 and subsequently by the Ethics Committees of the of other 19 Italian academic institutions, in which the study was run from December 2001 to present (study protocol registration number NCT00276929). The study protocol required that patients with primary eosinophilia (more than 1.5×10 eosinophils/L for more than 6 months) were screened for cytogenetic and molecular studies, including the FIP1L1-PDGFRα rearrangement, and were eligible for an experimental treatment with imatinib. Prior treatment was always discontinued for at least 15 days with the exception of corticosteroids, which were maintained at the same dose until a response to imatinib was shown and confirmed for 15 days. Imatinib was provided free-of-charge by Novartis Pharma (Origgio, Italy) in 100 mg tablets and was given in a single dose, beginning with 100 mg daily for one week. Thereafter, the daily dose was increased by 100 mg each week, and was set at 400 mg from the 4 week on. Imatinib treatment was continued for a minimum of 4 weeks in case of no hematologic response (HR), or until it was felt that it was beneficial for the patient, in case of response. During the first year, the dose was adjusted for toxicity and adverse events, according to the standard criteria for dose adjustment used in the treatment of chronic myeloid leukemia.32, 33 After 1 year of treatment, the dose could be adjusted at the investigator’s discretion. The patients were visited weekly for 1 month, the 12 monthly for 1 year, and every 3 months thereafter. Blood counts and differentials were performed at each visit. In patients with FIP1L1-PDGFRα rearrangement, the level of the transcripts was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) on marrow cells, every 3 months until confirmed negativity, and then every 6 months.

Three types of response were considered, hematologic, clinic, and molecular. A complete hematologic response (CHR) required a normal white blood cell count with a total eosinophil count of less than 0.250×10/L for more than 4 weeks. The HR was defined as partial (PHR) if the total eosinophil count was reduced to less than 50% of baseline value but was still higher than 0.250×10/L. The definition of clinical response was that all the symptoms, signs and laboratory evidence of organ and tissue involvement or dysfunction disappeared completely for more than 4 weeks. A complete molecular response required negativity of the RT-PCR for the FIP1L1-PDGFRα transcripts on two successive tests at a 3-month interval.

Laboratory tests

Conventional cytogenetic analysis was performed on bone marrow cells that were cultured without stimulation for 48 hours. Metaphases were G-banded with Wright’s stain. Chromosome abnormalities were classified according to the International System for Chromosome Nomenclature.34

Fluorescence in situ hybridization (FISH) was performed on interphase marrow cells, using a commercially available double fusion signal D-FISH BCR-ABL probe (Oncor, Appligene, Gaithesburg, MD, USA) to exclude the involvement of the BCR and ABL genes, a bacterial artificial chromosome (BAC) bk 350N15 spanning the fibroblast growth factor receptor 1 gene (FGFR1) (kindly provided by Dr. Negrini, University of Ferrara) for the 8p11 breakpoint. BAC probes (kindly provided by Dr. Rocchi, University of Bari) were used for the 5q33.1 platelet derived growth factor receptor beta (PDGFRβ) (RP 11–368O19), for the 4q12 PDGFRα (RP 11–231C18) and for the 4q12/CHIC2 (RP 11–3671N). BAC DNA was isolated from cultures using a standard miniprep procedure, and labeled with fluoroscein isothiocyanate or cyanine 3.5 by nick translation All FISH analyses were performed with a Nikon fluorescence microscope (Eclipse E1000, Nikon Instruments) attached to a computer-based imaging system (Genikon) equipped with a triple bandpass filter for 4,6-diamino-2-phenyl indole (DAPI), fluorescent isothiocyanate, and rhodamine.

For molecular studies, marrow mononuclear cells were obtained by Ficoll-Hypaque density gradient separation. Aliquots of 5×10 cells were resuspended in 600 μL GITC, and RNA was obtained following standard procedures. A qualitative RT-PCR was performed for BCR-FGFR1, BCR-ABL, and TEL-PDGFRβ, using standard conditions.35 For detection of FIP1L1-PDGFRα fusion transcripts, a nested RT-PCR was performed according to Cools et al.17 The fusion was analyzed using primers FIP1L1-F1 (5’-acctggtgctgatctttctgat) and PDGFRα-R1 (5’-tgagagcttgtttttcactgga) during the first PCR, and primers FIP1L1-F2 (5’-aaagaggatacgaatgggacttg) and PDGFRα-R2 (5’-gggaccggcttaatccatag) for the second PCR. In selected cases and for sequencing purposes, PCR products were cloned in pGEM-T-easy (Promega) and cloned products were sequenced on an ABI-PRISM 377 system (Perkin Elmer) according to manufacturer’s instructions.

Results

Patients

One hundred and ninety-six patients with eosinophilia were screened; 72 were identified as having primary or idiopathic eosinophilia, and 63 of these provided consent to study procedures and experimental treatment. Preliminary data of some of these patients were reported orally at the meetings of the European Haematology Association36 and the American Society of Hematology37, 38. The case of one patient was already published separately.24 The observation period of these 63 patients, from the first dose of imatinib, ranged between 15 and 60 months (median 25 months). Twenty-seven patients (43%) were found to carry the FIP1L1-PDGFRα rearrangement, while 36 (57%) were negative (Table 1). The main difference between patients with and without the rearrangement was gender, which was male in all 27 patients with the rearrangement vs 25 of 36 in negative cases. Other minor differences concerned age, with a median of 50 years in positive patients vs 58 years in negative ones; the total eosinophil count (median 4.8×10/L in positive patients vs 3.4×10/L in negative patients); skin involvement, which was recorded in only 5 patients without the rearrangement, and splenomegaly, which was reported in five patients with the rearrangement and in only one without. Prior duration of eosinophilia was also slightly different, the median being 16 months for patients with the rearrangement and 25 months for those without. Prior treatment, including corticosteroids, hydroxyurea or interferon-α, had been received by 66% of patients in both groups. All patients had a normal karyotype constitution with the exception of two patients without the rearrangement (45XY/45X, and 46XX+m) and one with the rearrangement (47XY+4 in 2 of 26 metaphases). All patients tested negative on FISH and RT-PCR for BCR-ABL, FGFR1-BCR, TEL-PDGFRβ, and cKit mutations.

Table 1.Clinical and hematologic characteristics of study patients, according to FIP1L1-PDGFRα rearrangement.

Treatment results

All 27 patients with FIP1L1-PDGFRα rearrangement achieved a CHR within 1 month and all 27 patients have remained in continuous CHR until last contact, for 15 to 60 months (median 25 months) (Table 2). Of the 15 patients with organ involvement, 13 achieved also a complete clinical remission, since all symptoms, signs and laboratory evidence of lung (5 cases), spleen (5 cases), liver (1 case) and soft tissue (2 cases) involvement disappeared within 3 months. In the remaining two patients, who had cardiac involvement, the echocardiographic patterns were not significantly modified after 12 and 30 months of treatment. All 27 patients became RT- PCR negative for the FIP1L1-PDGFRα fusion transcripts after 1 to 10 months of treatment (median 3 months). In 24 of them treatment has not been discontinued, and all these 24 patients have remained PCR-negative for 6 to 56 months (median 19 months). In three patients imatinib was discontinued after 12, 14 and 15 months of treatment. In all these 3 cases the FIP1L1-PDGFRα fusion transcripts became detectable again 4, 2, and 6 months after discontinuation of imatinib. In all three patients imatinib was reassumed at a dose of 200 mg daily, and the fusion transcripts disappeared after 2, 5, and 2 months of treatment.

Table 2.Hematologic response to imatinib treatment.

The mean daily dose of imatinib ranged from 100 to 386 mg (median 339 mg). All patients were still on treatment at last contact, at a daily dose of 400 mg (8 patients), 300 mg (2 patients), 200 mg (10 patients), and 100 mg (7 patients) (Table 3).

Table 3.Imatinib dose. As per protocol, all patients should have received 100 mg daily the first week, 200 mg daily the second week, 300 mg daily the third week, and 400 mg daily from the fourth week on, indefinitely in the case of response, or for 90 days in case of no response.

Four of 36 patients achieved a CHR in 1 month, and another patient in 3 months, for an overall CHR rate of 14% (5/36). All five patients who achieved CHR lost the response after 1, 1, 6, 9, and 15 months of imatinib treatment. Another four patients achieved a PHR, which was lost in less than 6 months. The clinical and hematologic characteristics of responding patients could not be distinguished from those of non-responding patients, but four of the complete responders were younger (18 to 38 years old). The daily dose of imatinib was similar to that taken by the patients with the rearrangement (Table 3), but the treatment duration was much shorter, since treatment was discontinued earlier because of lack of response, disease progression or response loss. At last contact, all 36 patients without the FIP1L1-PDGFRα rearrangement were alive with eosinophilia.

The type and frequency of grade 2 and 3 adverse events are reported in Table 4. Hematologic toxicity was negligible and limited to grade 2 neutropenia in 7.9% of cases and grade 3 neutropenia in 3.2% of cases. Thrombocytopenia greater than grade 1 was never reported. Non-hematologic adverse events consisted mainly of myalgia and muscle cramps (9.5% grade 2 and 3.1% grade 3) and diarrhea (6.3% grade 2), but also skin rash, abdominal pain, edema, headache and paresthesia (Table 4). In the group with the FIP1L1-PDGFRα rearrangement, treatment was not abandoned in any patient, but was discontinued temporarily in three patients for three times and in four patients once. In the group without the rearrangement, treatment was abandoned in two patients because of grade 3 skin rash, and in one patient because of grade 3 abdominal pain. No clinical evidence of cardiac dysfunction was reported.

Table 4.Frequency of all grade 2 and 3 adverse events. In FIP1L1-PDGFRα positive patients 61% of adverse events occurred during the first quarter of treatment, 23% during the second quarter and 16% after the first year of treatment.

Discussion

In a prospective multicenter study of eosinophilia we screened 196 patients with this condition and identified the cause of eosinophilia in 124. We selected the remaining 72 patients with primary or idiopathic eosinophilia and a total eosinophil count of more than 1.5×10/L for more than 6 months, and studied and treated 63 of them with imatinib. Thirty-six of the 63 study patients also had symptoms or signs of organ or tissue involvement or dysfunction. The study patients were treated with imatinib and a CHR was achieved rapidly in 27/27 of the patients with the rearrangement and in 5/36 of those without. The response was stable over the whole observation period (15 to 60 months, median 25 months) in all patients with the rearrangement, while all patients without the rearrangement lost their response in 1 to 15 months and discontinued imatinib treatment.

The high sensitivity to imatinib of HES/CEL with the FIP1L1-PDGFRα rearrangement has already been established. At least 47 cases had been reported, with an overall CHR rate of 95% (Table 5). Hoewever, many reports focused on the response rate and could not report on response duration. The maximum observation time after the first dose of imatinib was 24 months (Table 5). Now that the issue of the response rate has been settled, because it is increasingly clear that it is very close to 100%, the interest is focused on the quality, the stability, and the duration of the response. We found that with a median follow-up of 25 months, ranging from 15 to 60 months, the response is stable and durable in all patients carrying the rearrangement. Although molecular negativity is not equivalent to cure, it is the best available marker of the quality of the response. The achievement of molecular negativity was also reported in prior studies, but the stability and the duration of the negativity was not fully established.18, 21–23, 25, 29 We show that molecular negativity is durable and stable in all patients, but is dependent on treatment continuation, because molecular negativity was lost in three patients who discontinued imatinib and then regained when imatinib was resumed. Pending independent confirmation, this study suggests that the stability of the response requires continuation of imatinib treatment. This suggestion is supported by a recent update of imatinib treatment of myelodysplastic syndromes with the PDGFRβ-rearrangement, reporting that also in these cases the response was stable only if treatment was continued.39 In prior studies the daily dose of imatinib ranged between 100 and 600 mg, with the majority of the patients being treated with 100 or 200 mg.29, 30 In this study the planned daily dose was 400 mg, but actually ranged between 100 and 400 mg, and the majority of the patients continue treatment at a daily dose of 100 or 200 mg. Since imatinib inhibits PDGFRα at nanomolar concentration, an imatinib dose of 100 or 200 mg is likely to be sufficient to obtain and maintain the response. Moreover, there are reports of cases in which the response was maintained with 100 mg every second day, and even once a week.25, 31 The issue of the dose is important both because the cost of the drug, which may even create serious obstacles to treatment continuation,30 and because of the possible side effects. In these HES/CEL patients with the FIP1L1-PDGFRα rearrangement, imatinib was well tolerated (Table 4), and apparently even better than in patients with chronic myeloid leukemia. Hematologic toxicity was negligible at a dose of 400 mg daily, and non-hematologic toxicity was also limited, so that the overall compliance to treatment was excellent. However the chronic administration of a protein tyrosine kinase inhibitor may have effects difficult to predict. A recent report warned about cardiac toxicity,40 although cardiac adverse events had not been of concern in large series.41, 42 Moreover, chronic imatinib treatment may affect bone turn-over, and calcium and glucose metabolism.43, 44 It is therefore wise to keep the dose of imatinib at the minimum effective level and to carefully monitor the state of health of these highly selected patients. Before the FIP1L1-PDGFRα rearrangement was identified, several molecularly undefined HES patients were treated with imatinib and responded to this drug.11, 15 Clearly, these patients probably carried the rearrangement, but also patients without the rearrangement can respond to imatinib.17, 20–31 Previously, a documented CHR was reported in at least eight of 33 patients (24%) without the rearrangement (Table 5). The duration of the response was not reported for several of these patients. In our series, five of 36 (14%) patients without the rearrangement had a CHR, but all responders have relapsed on treatment. The bases for these responses are not clear, whether the target of imatinib was PDGFRα or other non-identified protein tyrosine kinases. From a clinical point of view, the indiscriminate use of imatinib or other protein tyrosine kinase inhibitors in cases in which a specific molecular target cannot be identified, cannot be recommended, but from a biological point of view the interest may be notable because of the possibility of discovering the role of other protein tyrosine kinases. Also of interest is the nearly absolute prevalence of male gender. Only two female patients have been shown to carry the rearrangement, so far.22, 28

Table 5.Summary of prior reports of cases of primary or idiopathic eosinophilia treated with imatinib. The reported maximum follow-up was 24 months, vs 15 to 60 months (median 25 months) in this study.

In conclusion, we have confirmed that imatinib is the treatment of choice for patients with FIP1L1-PDGFRα-rearranged HES/CEL, since almost all patients achieve and maintain complete hematologic, clinical and molecular remissions, chronic treatment is well tolerated, and responses are stable over time at doses as low as 100 mg daily.

Acknowledgments

Paolo Ricci (Bologna); Pier Ferruccio Ballerini (Conegliano); Filippo Gherlinzoni, Cristina Danesin and Cristina Tecchia (Treviso); Sergio Morandi (Cremona); Ercole De Biasi (Castelfranco Veneto); Paolo Vivaldi (Trento); Barbara Izzo (Naples); Pier Paolo Fattori (Rimini); Eliana Zuffa and Barbara Giannini (Ravenna); and Antonio Spadea (Rome) referred and cared for several patients. Their co-operation is gratefully acknowledged

Footnotes

• Authors’ contributions MB is the author taking primary responsability for the paper: GM, GS, FP, GR and FR promoted and designed the study; MR, SM, MT, FB and SP took care of the clinical part of the study; DC, FM, NT, EG, II, EO and SS performed cytogenetic and molecular studies; MR, AdV and CA collected and analyzed the data; MB and GM wrote the report, with the contribution of DC, MR and GS.
• Conflict of interest MB and GS have received reesearch grants and honoraries as speaker and consultant from Novartis Pharma; GR has received honoraries as speaker from Novartis Pharma; FP and GM have received research grants from Novartis Pharma; FR was an employee of Novartis Pharma; CA is an employee of Novartis Pharma.
• Funding: the study was supported by COFIN 2003 and Fondazione del Monte di Bologna e Ravenna grants.

• Received February 23, 2007.
• Accepted June 27, 2007.