The use of Imatinib in the treatment of Chronic Myelogenous Leukemia (CML) leads to the achievement of Complete Cytogenetic Response (CCR) in 70%-90% of patients, with 10%-30% showing persistence of Ph+ cells after standard (400 mg/day) or even high (600-800 mg/day) doses of the drug. These patients are thus cytogenetically resistant to Imatinib alone [1Kantarjian HM, Cortes JE, O'Brien S. Long-term survival benefit and improved complete cytogenetic and molecular res-ponse rates with Imatinib Mesylate in Philadelphia chromosome-positive chronic-phase chronic myeloid leukemia after failure of interferon-{alpha} Blood 2004; 104: 1979-88., 2O'Brien SG, Guilhot F, Larson RA. IRIS Investigators. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia N Engl J Med 2003; 348: 994-1004.].

Before the advent of 2^nd generation tyrosine-kinase (TK) inhibitors, treatment of resistant patients was uncertain and mainly based on the transplant procedures, unfortunately, in almost half of the cases, this approach was hampered by patient age or donor lack [3Hochhaus A, La Rosée P. Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance Leukemia 2004; 18: 1321-31.]. The association of Imatinib with other drugs, such as low-dose cytarabine, was tested in vitro [4Topaly J, Zeller WJ, Fruehauf S. Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukaemia cells Leukemia 2001; 15: 342-47.-6Tipping AJ, Mahon FX, Zafirides G. Drug responses of imatinib mesylate-resistant cells: synergism of imatinib with other chemotherapeutic drugs Leukemia 2002; 16: 2349-57.] and in vivo [7Gardembas M, Rousselot P, Tulliez M. Results of a prospective phase II study combining imatinib mesylate and cytarabine for the treatment of Philadelphia-positive chronic myelogenous leukemia patients in chronic phase Blood 2003; 102: 4298-305., 8Giles FJ, Feldman EJ, Roboz GJ. Phase II study of troxacitabine, a novel dioxolane nucleoside analog in patients with untreated or imatinib mesylate-resistant chronic myelogenous leukemia in blastic phase Leuk Res 2003; 27: 1091-96.] as alternative therapy, but results were conflicting.

Hydroxyurea (HU) has been employed in CML setting for more than 40 years and its efficacy in managing leukocytosis as well as other disease-related symptoms is well known; in addition, its toxicity profile is very favorable.

Thus, before the introduction of new TK inhibitors, we considered HU a good candidate to be added to Imatinib in patients resistant to this drug alone and report herein our experience on this association.

Imatinib resistance in patients, receiving the drug at the standard dosage of 400 mg/day was defined according to the following criteria:

1. lack of any cytogenetic response after 6 months, or
2. lack of major cytogenetic response (MCR) after 12 months

Cytogenetic analyses were performed on bone marrow (BM) aspirates from direct or short term (24-48h) cultures, with or without colcemid exposure. These had been carried out at diagnosis, before starting imatinib, after 3, 6, and 12 months of therapy with Imatinib, before adding HU, after 3, 6 and 12 months of HU and Imatinib association and thereafter every six months. Metaphases were examined after GAG banding, according to standard methods. Cytogenetic response was categorized according to the standard criteria. CCR was defined as the presence of 100% Ph- metaphases, MCR as the presence of > 66% Ph- metaphases.

Qualitative RT-nested PCR was carried out according to the standardized RT-PCR analysis of fusion gene transcripts, as previously described in the report of the BIOMED-16 [9Dongen JJ, Macintyre EA, Gabert JA. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease Leukemia 1999; 13: 1901-28.].

Imatinib has profoundly changed the prognosis of CML, and at present the vast majority of patients can achieve a CCR and possibly a complete disappearance of BCR/ABL hybrid gene, however, patients who do not achieve CCR after 12-18 months of Imatinib treatment must be considered resistant and are candidate to alternative therapies.

An international panel of experts recently published general guidelines for defining resistance to Imatinib [10Baccarani M, Saglio G, Goldman J. Evolving concepts in the management of chronic myeloid leukemia Recommendations from an expert panel on behalf of the European Leukemianet Blood 2006; 108: 1809-.]. These guidelines are now worldwide accepted, but they were not available when resistance to Imatinib was considered in the present study; nevertheless, all the patients included would be classified as resistant to Imatinib also according to the present definition of resistance.

Along all this study period, new TK inhibitors (nilotinib and dasatinib) were not yet available for treatment of Imatinib resistant patients, the only chance we had at that time for patients not eligible for transplant procedures was the association with other “old” drugs, and it is conceivable that many resistant patients have been managed in such way. Some previous in vitro studies have shown conflicting results on reciprocal effects between Imatinib and HU [4Topaly J, Zeller WJ, Fruehauf S. Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukaemia cells Leukemia 2001; 15: 342-47.-6Tipping AJ, Mahon FX, Zafirides G. Drug responses of imatinib mesylate-resistant cells: synergism of imatinib with other chemotherapeutic drugs Leukemia 2002; 16: 2349-57.], however, HU seemed us to be a reasonable association mainly due to its very low toxicity profile as compared with other drugs (i.e. low-dose cytarabine).

Our results in this small Imatinib resistant patient population show that association of HU and Imatinib is capable to induce a sustained cytogenetic response in at least one half of patients resistant to Imatinib alone, with minimal toxicity. The duration of such responses appears often short, but 2/7 of our patients achieved a long-lasting (> 12 months) CCR.

Mutational status of our patients was not routinely studied before HU addition, because in that period its role and significance were still unclear. As a matter of fact, only 5/11 patients were assessed for point mutations before HU addition, however, it is worth to note that a patient with F317L mutation, which is at present known to be resistant to Dasatinib [11Soverini S, Martinelli G, Colarossi S. Presence or the emergence of a F317L BCR-ABL mutation may be associated with resistance to dasatinib in Philadelphia chromosome-positive leukemia J Clin Oncol 2006; 24: e51-2.], achieved a relatively long response with this association.

We are obviously aware that new TK inhibitors have a major role as salvage treatment in CML patients primarily resistant to Imatinib. However, there are at least 2 subsets of patients for which the addition of HU to Imatinib could represent a useful additional choice:

very elderly patients (aged > 75 - 80 years) as 1^st line treatment, as Imatinib very often must be employed at dosage < 400 mg/day and the association of HU could improve response rate without adding severe toxicity.

patients resistant/intolerant to new TK inhibitors and not eligible for transplant procedures as 3^rd line treatment.

Moreover, to the best of our knowledge, this study also provides the first in vivo evidence of an additive activity of HU and Imatinib, this evidence could suggest to combine HU and new TK inhibitors in patients resistant to new drugs given alone, particularly to face with some mutations known to be unresponsive to other approaches.