Gunnar Birgegård
email: gunnar.birgegard@medsci.uu.se

Long-term management of thrombocytosis in essential thrombocythaemia

Keywords: Essential thrombocythaemia, Anagrelide, Hydroxycarbamide, Interferon, Myeloproliferative disorder

Essential thrombocythaemia (ET) is an acquired myeloproliferative disorder with a prolonged clinical course and a near-normal life expectancy. Therapy is stratified according to risk of thrombohaemorrhagic events. In highrisk patients, platelet reduction is generally recommended. In intermediate-risk patients, therapy should be considered depending on the severity of associated risk factors, especially cardiovascular. In low-risk patients, a watchand-wait approach is appropriate. Hydroxycarbamide is generally first-line therapy. Concerns for possible leukemogenicity make anagrelide or interferon-α possible choices in younger patients and those who are resistant or intolerant to hydroxycarbamide. Each pharmacotherapy is associated with specific long-term risks and benefits. The potential risk of major bleeding is the main drawback of aspirin. Hydroxycarbamide is an established, effective drug for ET, but it may increase the risk of transformation to acute myeloid leukaemia and may give mucocutaneous ulcers. Anagrelide is a licensed treatment that also reduces platelet counts and is generally well tolerated, with evidence that some common side effects diminish over time. Anagrelide can have cardiac effects due to inhibition of phosphodiesterase III and therefore requires cautious use in patients with cardiac insufficiency. There is no evidence of leukaemogenicity with anagrelide or interferon-α therapy. Interferon-α is the only treatment suitable for use during pregnancy, although it is not licensed in ET. While it is effective for platelet reduction, the use of interferon-α is restricted by psychiatric side effects. Our knowledge of the optimum pharmacotherapy for each patient with ET continues to evolve through research and clinical trials, particularly into the molecular basis of the disease.

Introduction

Essential thrombocythaemia (ET) is an acquired myeloproliferative disorder (MPD), characterised by persistent peripheral thrombocytosis and a tendency for thrombosis and haemorrhage [START_REF] Briere | Essential thrombocythemia[END_REF]. For example, in a study of 93 patients with ET, at a median follow-up of 70 months, 16% of patients developed thrombosis, 14% developed haemorrhage and 17% developed microvascular complications [START_REF] Jensen | Incidence, clinical features and outcome of essential thrombocythaemia in a well defined geographical area[END_REF]. ET can proceed in some cases to myelofibrosis, acute myeloid leukaemia (AML), myelodysplastic syndromes (MDS) or polycythaemia vera (PV) [START_REF] Campbell | Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study[END_REF]. The estimated annual incidence of ET based on World Health Organization (WHO) criteria is 1-2.5/100,000 individuals [START_REF] Sanchez | Essential thrombocythemia: a review of diagnostic and pathologic features[END_REF]. However, the true incidence is likely to be higher as many patients are without symptoms, and thus undiagnosed. Even though ET is a rare disease, the prevalence is high at 30/ 100,000 general population [START_REF] Briere | Essential thrombocythemia[END_REF], reflecting the near-normal life expectancy of patients with the condition [START_REF] Penninga | Polycythaemia vera and essential thrombocythaemia: current treatment strategies[END_REF].

Several lines of evidence suggest that ET is a heterogeneous disease entity, though the existence of distinct subgroups is difficult to validate. Molecular evidence includes the patient's JAK2 mutation status (i.e. the presence or absence of the Janus kinase 2 mutation JAK2V617F) [START_REF] Campbell | Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study[END_REF], variation in X-chromosome inactivation patterns in females [START_REF] Harrison | A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications[END_REF] and differences in PV rubra vera-1 (PRV1) mRNA expression (discriminating two types of ET) [START_REF] Griesshammer | PRV-1 mRNA expression discriminates two types of essential thrombocythemia[END_REF], while immunohistochemical evidence includes heterogeneous c-mpl expression [START_REF] Teofili | The expression pattern of c-mpl in megakaryocytes correlates with thrombotic risk in essential thrombocythemia[END_REF]. Differences have also been observed between patients with ET in the growth of erythropoietin-independent colonies (EEC). In a study of molecular markers, growth of EEC was observed in 50-69% of ET patients [START_REF] Griesshammer | PRV-1 mRNA expression discriminates two types of essential thrombocythemia[END_REF][START_REF] Kralovics | Comparison of molecular markers in a cohort of patients with chronic myeloproliferative disorders[END_REF]. Furthermore, as a group, ET patients with the JAK2V617F mutation and/or low serum erythropoietin levels seem to have a more PV-like phenotype with slightly higher white blood cell counts and haemoglobin levels [START_REF] Campbell | Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study[END_REF][START_REF] Carobbio | Leukocytosis is a risk factor for or hydroxyurea[END_REF]11].

Diagnosis

The diagnosis of ET includes detection of specific changes in bone marrow morphology, as well as exclusion of secondary thrombocytosis caused by other conditions [12]. Recently, an advisory group to the WHO proposed revised diagnostic criteria for ET (Table 1) [12]. Using these criteria, diagnosis is supported by demonstration of a clonal marker of the condition (e.g. JAK2V617F, present in approximately 50% of ET patients) [START_REF] Campbell | Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study[END_REF]; however, identification of this mutation cannot differentiate between ET and other MPDs. Several investigators suggest that some ET patients diagnosed according to the guidelines of the Polycythaemia Vera Study Group actually have an early stage chronic primary myelofibrosis (PMF-0 or 'false ET') [13,14]. This prefibrotic condition is known to progress to overt myelofibrosis in a high proportion of patients, thus differing from 'true ET' which does not [15][16][17]. If this distinction is valid, it is an important consideration when evaluating treatment strategies. However, the feasibility of distinguishing ET from PMF-0 is not generally accepted among pathologists in clinical practice [18].

The ideal therapeutic agent?

A number of pharmacotherapies have been investigated for use in ET. The ideal therapeutic treatment should rapidly lower platelet counts to normal levels, reduce ET-related complications and symptoms, have limited side effects and no long-term safety concerns. Long-term benefits and risks of pharmacotherapy are of particular relevance to patients with ET, who require prolonged treatment over a lifetime. This article considers the long-term effects of several ET treatment options, focusing on the anti-platelet agent, aspirin, and three cytoreductive therapies: hydroxycarbamide, anagrelide and interferon-α. Taking these findings into consideration, a risk-based treatment algorithm for ET is proposed.

Literature search

This article contains information from a number of sources, including relevant searches of PubMed (conducted on 14 November 2007), the author's own reference database and the reference lists of recent review articles. It is not intended as a systematic review.

Acetylsalicyclic acid (aspirin)

Since it was first synthesised in 1897, the use of aspirin has extended from analgesia to encompass prevention of cardiovascular events such as myocardial infarction and ischaemic stroke [19]. Theoretically, aspirin also has a place in the management of ET, particularly as it has been shown to suppress excess thromboxane synthesis [20], evident in patients with ET and PV [20,21]. Several studies have confirmed this theory, showing that aspirin reduces thrombotic complications associated with ET [22,23] as well as microvascular events (e.g. neurological symptoms and erythromelalgia) [24,25].

The only randomised controlled trial investigating use of aspirin in this area was performed in patients with PV, a related MPD [26], which calls into question the direct applicability of the results. In the placebo-controlled European Collaboration on Low-dose Aspirin in PV (ECLAP) study, low-dose aspirin (100 mg/day) significantly reduced the risk of thrombotic complications (non-fatal myocardial infarction and stroke, death from cardiovascular causes, pulmonary embolism and major venous thrombosis) [26].

Any long-term therapeutic benefit of aspirin should be weighed against the potential risk of a major bleeding episode and gastrointestinal side effects. The relative risk of major bleeding with aspirin was 1.62 in the ECLAP study, which was consistent with results in non-PV patients [26]. Noteworthy is that about 70% of patients in both groups also received antiplatelet prophylaxis. The United Kingdom Medical Research Council Primary Thrombocythaemia 1 study (MRC PT-1) compared hydroxycarbamide plus aspirin (n=404) with anagrelide plus aspirin (n=405) [30]. Rates of major arterial and venous thrombosis in the hydroxycarbamide arm (4.2% and 3.5%, respectively) were similar to those observed in the hydroxycarbamide group of the Cortelazzo study [29,30]. The incidence of venous thrombosis was significantly lower in the anagrelide arm than in the hydroxycarbamide arm (p=0.006), whereas the incidence of arterial thrombosis was lower in the hydroxycarbamide arm (p=0.004). However, the reasons behind these observations are unclear. Five patients developed myelofibrosis in the hydroxycarbamide group versus 16 in the anagrelide group; raising the possibility that hydroxycarbamide may give better protection against fibrosis. Significantly fewer haemorrhagic events occurred in the hydroxycarbamide group than in the anagrelide group, 8 versus 22, respectively (p<0.008). The latter finding may be due to a synergistic effect between anagrelide and aspirin when given concomitantly. No significant difference in the frequency of minor bleedings between treatment groups was observed [30]. It is noteworthy that a separate randomised controlled investigation (ANAHYDRET study) compared hydroxycarbamide and anagrelide in 258 previously untreated ET patients diagnosed according to the WHO criteria [34]. The 12-month findings, reported at the American Society of Hematology congress in 2007, showed no significant difference in the incidence of thromboembolic or haemorrhagic events (major events 8 versus 8 and minor events 22 versus 23 for thromboembolic and haemorrhagic events, respectively) between the hydroxycarbamide and anagrelide arms, suggesting noninferiority for anagrelide. However, statistically low numbers preclude a firm conclusion at this timepoint [34].

There is strong evidence to suggest that concomitant or sequential use of hydroxycarbamide and an alkylating agent or radioactive phosphorous may increase the risk of malignant transformation in MPDs, possibly due to the radiosensitising effect of hydroxycarbamide [35][36][37][38][39]. This theory is supported by mechanism of action studies, which show that hydroxycarbamide prevents de novo DNA synthesis and repair [40]. Interestingly, the bone marrow of hydroxycarbamide-treated patients shows a dysplastic appearance, including left-shifting of myelopoiesis, macrocytosis of red cells and dysplasia of megakaryocytes, which is not seen with non-cytostatic drugs [41]. In clinical studies with more than 96 months median follow-up, transformation to AML/MDS occurred in 0-22% of patients with ET/PV treated with hydroxycarbamide as the sole therapeutic agent [33,37,[42][START_REF] Chim | Long-term outcome of 231 patients with essential thrombocythemia: prognostic factors for thrombosis, bleeding, myelofibrosis[END_REF][START_REF] Fruchtman | From efficacy to safety: a Polycythemia Vera Study group report on hydroxyurea in patients with polycythemia vera[END_REF][START_REF] Kiladjian | Long-term incidence of hematological evolution in three French prospective studies of hydroxyurea and pipobroman in polycythemia vera and essential thrombocythemia[END_REF][START_REF] Najean | Treatment of polycythemia vera: the use of hydroxyurea and pipobroman in 292 patients under the age of 65 years[END_REF]. The only long-term study designed to investigate this endpoint shows that transformation to AML in hydroxycarbamide-or pipobroman-treated patients generally occurs after 10 or more years' therapy, with the incidence reaching 10-12% after 12 years [START_REF] Kiladjian | Long-term incidence of hematological evolution in three French prospective studies of hydroxyurea and pipobroman in polycythemia vera and essential thrombocythemia[END_REF]. In conclusion, the leukaemogenicity of hydroxycarbamide remains a concern that has not been diminished by recent data.

The most common side effects of hydroxycarbamide are granulocytopaenia and anaemia [START_REF] Stevens | Hydroxyurea: an overview[END_REF], especially after longterm treatment. Fever of unknown aetiology has also been observed following initiation of hydroxycarbamide therapy [START_REF] Lossos | Hydroxyurea-induced fever: case report and review of the literature[END_REF][START_REF] Braester | Hydroxyurea as a cause of drug fever[END_REF][START_REF] Lannemyr | Hydroxyurea as a cause of drug fever in essential thrombocythaemia[END_REF]. Cutaneous manifestations are commonly reported by patients receiving long-term hydroxycarbamide therapy and are the most frequent reason for treatment cessation [START_REF] Kennedy | Skin changes secondary to hydroxyurea therapy[END_REF][START_REF] Dumont-Wallon | Effets secondaires cutanéomuqueux de l'hydroxyurée: étude prospective sur 27 patients[END_REF][START_REF] Zaccaria | Secondary cutaneous effects of hydroxyurea: possible pathogenetic mechanisms[END_REF]. Cutaneous complications include alopecia, xerosis and scaling, atrophy of the skin and subcutaneous tissues, skin and nail hyperpigmentation, and cutaneous and mucocutaneous ulceration [START_REF] Kennedy | Skin changes secondary to hydroxyurea therapy[END_REF][START_REF] Dumont-Wallon | Effets secondaires cutanéomuqueux de l'hydroxyurée: étude prospective sur 27 patients[END_REF]. Furthermore, an association between long-term hydroxycarbamide therapy and multiple skin tumours, including squamous and basal cell carcinomas, has also been reported [START_REF] Best | Multiple skin cancers associated with hydroxyurea therapy[END_REF]. The lower-limb ulcers caused by hydroxycarbamide are often painful, and treatment must be withheld to allow the ulcers to heal, but this occurs slowly and poorly, occasionally requiring grafting [START_REF] Ravandi-Kashani | Cutaneous ulcers associated with hydroxyurea therapy in myeloproliferative disorders[END_REF][START_REF] Gilbert | Modern treatment strategies in polycythemia vera[END_REF]. In one study, 12 out of 133 patients (9.0%) receiving hydroxycarbamide developed leg ulcers, requiring a switch of treatment in ten of these patients [START_REF] Najean | Treatment of polycythemia vera: the use of hydroxyurea and pipobroman in 292 patients under the age of 65 years[END_REF]. Withdrawal rates of 8-20% due to adverse effects have been reported in trials [30][31][32][START_REF] Najean | Treatment of polycythemia vera: the use of hydroxyurea and pipobroman in 292 patients under the age of 65 years[END_REF]. According to experts in the field [START_REF] Barbui | When and how to treat essential thrombocythemia[END_REF][START_REF] Dingli | A critical review of anagrelide therapy in essential thrombocythemia and related disorders[END_REF] and evidence-based guidelines [START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF], hydroxycarbamide is considered to be the first-choice cytoreductive therapy in high-risk patients with ET. However, approximately 15% of patients receiving hydroxycarbamide either do not achieve the desired reduction in platelet counts or develop unacceptable side effects [30-33, 46, 60]. In an attempt to standardise the definition of resistance and intolerance to hydroxycarbamide, thus when to stop treatment and switch to an alternative therapy, a working group consensus definition has recently been published (Table 2) [START_REF] Barosi | A unified definition of clinical resistance/ intolerance to hydroxyurea in essential thrombocythemia: results of a consensus process by an international working group[END_REF].

Anagrelide

Anagrelide is a cytoreductive agent, with a selective effect on the megakaryocyte cell lineage [START_REF] Hong | Comparison between anagrelide and hydroxycarbamide in their activities against haematopoietic progenitor cell growth and differentiation: selectivity of anagrelide for the megakaryocytic lineage[END_REF]. It reduces platelet production by inhibiting megakaryocyte colony development, thus reducing megakaryocyte size, ploidy and maturation [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF]. Several studies have shown that it effectively lowers platelet counts (overall response rate of 76-94%) and reduces ET-associated complications and symptoms [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF][START_REF] Fruchtman | Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders[END_REF][START_REF] Steurer | Anagrelide for thrombocytosis in myeloproliferative disorders: a prospective study to assess efficacy and adverse event profile[END_REF][START_REF] Penninga | Anagrelide treatment in 52 patients with chronic myeloproliferative diseases[END_REF][START_REF] Storen | Long-term use of anagrelide in young patients with essential thrombocythemia[END_REF][START_REF] Laguna | Effectiveness of anagrelide in the treatment of symptomatic patients with essential thrombocythemia[END_REF][START_REF] Mills | Efficacy, safety and tolerability of anagrelide in the treatment of essential thrombocythaemia[END_REF][70][START_REF] Study | Anagrelide, a therapy for thrombocythemic states: experience in 577 patients[END_REF][START_REF] Petitt | Anagrelide for control of thrombocythemia in polycythemia and other myeloproliferative disorders[END_REF]. Anagrelide has been shown in trials to be as effective as hydroxycarbamide, interferon-α and alkylating agents in reducing platelet counts, without the added complication of alkylating or cytotoxic properties [START_REF] Mazzucconi | Therapy with anagrelide in patients affected by essential thrombocythemia: preliminary results[END_REF], and can be used in patients who are intolerant or resistant to these cytoreductive agents [START_REF] Fruchtman | Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders[END_REF].

Anagrelide is not mutagenic [START_REF] Petrides | Anagrelide: a decade of clinical experience with its use for the treatment of primary thrombocythaemia[END_REF], and there is no known evidence to suggest that it is leukaemogenic [START_REF] Barbui | The leukemia controversy in myeloproliferative disorders: is it a natural progression of disease, a secondary sequela of therapy, or a combination of both[END_REF]. In a longterm retrospective analysis, anagrelide did not result in excess conversion to AML, and no ET patient receiving anagrelide for more than 3 years transformed [START_REF] Fruchtman | Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders[END_REF]. Moreover, in a study with a mean follow-up of 12.5 years, leukaemic transformation did not occur in any of the 39 ET patients treated with anagrelide [START_REF] Mazzucconi | A long-term study of young patients with essential thrombocythemia treated with anagrelide[END_REF].

Anagrelide is considered effective for reducing the incidence of thromboembolic and haemorrhagic complications. However, this effect has not been investigated in a placebo-controlled study. Supporting evidence rests mainly on the comparison with hydroxycarbamide; in particular, the study by Cortelazzo et al. [29] showing a reduction in thrombotic events with cytoreductive treatment, and the results of the PT-1 study [30], demonstrating a similar incidence of thrombotic events in the anagrelide and hydroxycarbamide arms to that reported in the Cortelazzo study. These two studies differed in terms of inclusion criteria and the use of aspirin; however, the incidence of thrombosis was the same in both hydroxycarbamide arms, indicating similarity of the population. The ANAHYDRET study demonstrated non-inferiority of anagrelide (for efficacy and ET-related complications) compared with hydroxycarbamide for the treatment of ET patients at 12 months follow-up (p<0.025) [34]. Furthermore, in a study of 79 patients with ET, anagrelide significantly reduced the rate of major thromboembolic complications compared with the pre-treatment period (p=0.0455) [START_REF] Steurer | Anagrelide for thrombocytosis in myeloproliferative disorders: a prospective study to assess efficacy and adverse event profile[END_REF]. However, a clear clinical benefit in terms of haemorrhagic events has not yet been convincingly demonstrated.

The most common side effects of anagrelide include headache (13-35%) and tachycardia (9-21%), which arise due to the inhibitory properties of anagrelide on phosphodiesterase III [START_REF] Steurer | Anagrelide for thrombocytosis in myeloproliferative disorders: a prospective study to assess efficacy and adverse event profile[END_REF][START_REF] Penninga | Anagrelide treatment in 52 patients with chronic myeloproliferative diseases[END_REF][START_REF] Mills | Efficacy, safety and tolerability of anagrelide in the treatment of essential thrombocythaemia[END_REF]70,[START_REF] Mazzucconi | Therapy with anagrelide in patients affected by essential thrombocythemia: preliminary results[END_REF][START_REF] Zomas | The use anagrelide for control of thrombocytosis in Greek patients with myeloproliferative disorders[END_REF]. The majority of common side effects are mild or moderate (e.g. WHO grade 1 or 2) and manageable [63-66, 70, 72, 77], with the frequency and severity dose-dependent [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF]. The incidence of serious side effects is higher in patients aged 60 years or older than in patients younger than 60 years [START_REF] Fruchtman | Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders[END_REF]. Aggravation of cardiac insufficiency is a rare but important side effect, and special caution is advised in patients with previous cardiac failure [START_REF] Mazzucconi | A long-term study of young patients with essential thrombocythemia treated with anagrelide[END_REF][START_REF] Birgegard | Anagrelide treatment in myeloproliferative disorders[END_REF]. Anagrelide lowers red blood cell count by approximately 10% in about 30% of patients [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF][START_REF] Gilbert | Diagnosis and treatment of thrombocythemia in myeloproliferative disorders[END_REF], thought to be due to its well-known vasodilatory properties [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF]. Anaemia was detected in about 50% of patients in a study by Penninga et al. but was mild and not clinically apparent in most [START_REF] Penninga | Anagrelide treatment in 52 patients with chronic myeloproliferative diseases[END_REF]. Lowgrade anaemia was shown to persist during a 2-year followup study, but not to progress.

Appropriate management of side effects may lead to successful long-term acceptance of medication. In several studies, the majority of side effects with anagrelide occurred within 1 month of starting therapy [START_REF] Steurer | Anagrelide for thrombocytosis in myeloproliferative disorders: a prospective study to assess efficacy and adverse event profile[END_REF][START_REF] Penninga | Anagrelide treatment in 52 patients with chronic myeloproliferative diseases[END_REF][START_REF] Laguna | Effectiveness of anagrelide in the treatment of symptomatic patients with essential thrombocythemia[END_REF]70,[START_REF] Study | Anagrelide, a therapy for thrombocythemic states: experience in 577 patients[END_REF], with evidence showing that they subsided during continued treatment [65-71, 77, 80]. This effect is clearly Resistance/intolerance to hydroxycarbamide requires fulfilment of at least one of the following criteria: Platelet count of: >600,000/μl after 3 months of treatment with at least 2 g/day hydroxycarbamide a >400,000/μl and leukocytes <2,500/μl at any dose of hydroxycarbamide >400,000/μl and haemoglobin <10 g/dl at any dose of hydroxycarbamide Presence of leg ulcers or other unacceptable mucocutaneous manifestations at any dose of hydroxycarbamide Fever related to hydroxycarbamide a 2.5 g/day in patients with a body mass of >80 kg illustrated in the study by Storen and Tefferi involving 35 patients younger than 50 years ET [START_REF] Storen | Long-term use of anagrelide in young patients with essential thrombocythemia[END_REF]. Comparing initial (less than 3 months) and longer-term (over 3 months) anagrelide treatment, the incidence of headache was reduced from 34.2% to 5.7%, tachycardia from 22.8% to 8.5%, oedema from 14.2% to 5.7% and diarrhoea from 8.5% to 0% [START_REF] Storen | Long-term use of anagrelide in young patients with essential thrombocythemia[END_REF]. In contrast, a study by Birgegard et al. showed that the intensity of side effects remained the same in some patients with long-term treatment [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF]. These findings suggest that it may be beneficial to inform patients of the possible reduction in side effects with time in order to improve compliance, especially in the early stages of treatment. Alternatively, it is feasible that this observed reduction was due to the withdrawal of patients who cannot tolerate anagrelide, hence only those patients experiencing milder side effects were recorded. However, in the study by Storen and Tefferi, no patients were lost to follow-up; therefore, it appears that the reduction in side effects might be related to improved tolerance to treatment with time.

For some ET patients, side effects can be prolonged and may lead to discontinuation of therapy: 0-50% of patients were withdrawn from anagrelide treatment in published studies [START_REF] Birgegard | Adverse effects and benefits of two years of anagrelide treatment for thrombocythemia in chronic myeloproliferative disorders[END_REF][START_REF] Fruchtman | Anagrelide: analysis of long-term efficacy, safety and leukemogenic potential in myeloproliferative disorders[END_REF][START_REF] Laguna | Effectiveness of anagrelide in the treatment of symptomatic patients with essential thrombocythemia[END_REF][START_REF] Mills | Efficacy, safety and tolerability of anagrelide in the treatment of essential thrombocythaemia[END_REF]80], some because of lack of response and others because increasing the dose to an effective level was hindered by side effects.

Interferon-α

The interferon cytokine family demonstrates a wide range of biological properties, such as immune activation, as well as anti-proliferative and cell differentiation effects [START_REF] Tefferi | Treatment of polycythaemia vera and essential thrombocythaemia[END_REF]. Based on these properties, interferon-α was first investigated for the treatment of ET in the late 1980s; however, it has never received an EU licence for a platelet reduction indication. The mechanism of action of interferon-α is thought to relate to its anti-proliferative effects on megakaryocytes, with reduction in platelet half-life also a contributing factor [START_REF] Wadenvik | The effect of alpha-interferon on bone marrow megakaryocytes and platelet production rate in essential thrombocythemia[END_REF].

Interferon-α is effective in reducing platelet counts, with a mean overall response rate of 84.6% [START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF]. There has been no randomised comparison of interferon-α with other conventional therapies. Nevertheless, interferon-α is used off-label as a platelet-reducing alternative agent in MPDs, and is the only drug suitable for use during pregnancy [START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF]. It is of note that interferon-α is not known to be leukaemogenic [START_REF] Barbui | The leukemia controversy in myeloproliferative disorders: is it a natural progression of disease, a secondary sequela of therapy, or a combination of both[END_REF].

Recent results in patients with PV indicate that interferon-α can reduce the percentage of cells expressing the JAK2 mutant allele (i.e. the JAK2V617F allele burden) [START_REF] Kiladjian | High molecular response rate of polycythemia vera patients treated with pegylated interferon alpha-2a[END_REF][START_REF] Samuelsson | Limited effects on JAK2 mutational status after pegylated interferon alpha-2b therapy in polycythemia vera and essential thrombocythemia[END_REF][START_REF] Jones | Minimal molecular response in polycythemia vera patients treated with imatinib or interferon alpha[END_REF]. The biological and clinical significance of these results are unclear, and larger-scale studies are required to confirm the original findings. To date, no similar results have been published in patients with ET.

Common side effects of interferon-α include influenzalike symptoms, nausea, diarrhoea, myalgia, depression and fatigue [START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF][START_REF] Seewann | Interferon therapy in essential thrombocythemia[END_REF]. Side effects occur in nearly all patients at the start of treatment, but they generally abate [START_REF] Barbui | Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation[END_REF]. For example, in the study by Yataganas et al. investigating the use of interferon-α in 18 patients with an MPD (nine with ET), influenza-like symptoms (e.g. fever higher than 38°C) lasted a maximum of 4-8 days following treatment initiation [START_REF] Yataganas | alpha Interferon treatment of essential thrombocythaemia and other myeloproliferative disorders with excessive thrombocytosis[END_REF]. Despite this abatement, dropout rates between 15% and 66% have been reported, most commonly around 25% [START_REF] Samuelsson | A phase II trial of pegylated interferon alpha-2b therapy for polycythemia vera and essential thrombocythemia: feasibility, clinical and biologic effects, and impact on quality of life[END_REF][START_REF] Lengfelder | Interferonalpha in the treatment of essential thrombocythemia[END_REF][START_REF] Lengfelder | Interferon alpha in the treatment of polycythemia vera[END_REF].

Given its immunomodulatory properties, interferon-α may result in symptomatic episodes of autoimmune disease such as hypothyroidism [START_REF] Gilbert | Other secondary sequelae of treatments for myeloproliferative disorders[END_REF]. There have been reports of autoimmune thyroiditis [START_REF] Gisslinger | Interferon in essential thrombocythaemia[END_REF], arthritis [START_REF] Çabuk | Arthritis induced by interferon-alpha therapy in a patient with essential thrombocythemia[END_REF], hypertriglyceridaemia [START_REF] Salvo | Hypertriglyceridemia following treatment with interferon alpha in essential thrombocythemia[END_REF] and ischaemic optic neuropathy [START_REF] Vardizer | Interferon-alpha-associated bilateral simultaneous ischemic optic neuropathy[END_REF]. Mood disorders (depression/mania) are a well-documented side effect of interferon-α treatment [START_REF] Merup | Symptoms, symptom distress and health-related quality of life in patients with polycythaemia vera or essential thrombocythaemia during treatment with interferonalpha[END_REF][START_REF] Malek-Ahmadi | Neuropsychiatric complications of interferons: classification, neurochemical bases, and management[END_REF][START_REF] Malek-Ahmadi | Mood disorders associated with interferon treatment: theoretical and practical considerations[END_REF], and limit the clinical utility of this agent in the treatment of ET.

The use of pegylated-interferon-α has also been investigated for the treatment of MPDs. This agent is formulated from interferon-α by attaching high molecular weight polymers of ethylene glycol [START_REF] Quintas-Cardama | Pegylated interferon therapy for patients with Philadelphia chromosome-negative myeloproliferative disorders[END_REF]. Such chemical modification increases serum half-life and reduces renal excretion, thus allowing weekly, rather than daily, administration. Pegylated-interferon-α has been shown to effectively control platelet count and disease symptoms in patients with MPDs, including ET [START_REF] Samuelsson | A phase II trial of pegylated interferon alpha-2b therapy for polycythemia vera and essential thrombocythemia: feasibility, clinical and biologic effects, and impact on quality of life[END_REF][100][101][102]]. However, in two studies of 38 and 42 patients with an MPD, pegylatedinterferon-α had a similar tolerability profile to conventional interferon-α, with 26% and 50% of patients stopping therapy due to side effects and the level of toxicity limiting the duration of therapy [101,102].

Risk stratification for treatment management

ET is a chronic condition with a prolonged clinical course that cannot currently be cured. Management, therefore, relies on the prevention of serious complications, specifically thrombohaemorrhagic events, through reduction of the platelet count or by altering platelet function. Management is stratified according to risk, categorised as high, intermediate and low, though the exact definition varies by investigator and country. In addition to traditional risk factors, which include previous thrombosis or major bleeding, age and elevated platelet count, other less traditional risk factors have also been suggested that may influence treatment decisions. They include cardiovascular risk factors and molecular markers (e.g. presence of PRV-1 or JAK2 mutation) [103]. Whether the JAKV2617F mutation should be part of the risk stratification remains an area of uncertainty. Although evidence on whether the JAK2V617F mutation increases the risk of thrombotic complications is conflicting [104-108], overall it appears that having the mutation is associated with a higher risk. Some studies have indicated an association between leukocytosis and increased risk of thrombosis [START_REF] Carobbio | Leukocytosis is a risk factor for or hydroxyurea[END_REF]109,110], and one study showed in a time-dependent analysis that the increased risk was corrected by hydroxycarbamide treatment [START_REF] Carobbio | Leukocytosis is a risk factor for or hydroxyurea[END_REF]. It is possible that the leukocyte count is a prognostic marker for the activity of the MPD, but it remains to be proven by further study whether it is an important marker for treatment efficacy. The evidence for raised leukocyte levels being a risk factor for survival in ET is growing, but intervention studies are needed before white blood cell counts can be a basis for treatment decisions.

Taking into account the available evidence and clinical experience for various ET treatments, a suggested treatment algorithm is presented in Fig. 1. The following risk factors are suggested to define high-risk ET patients: (1) previous thrombosis or major bleeding; (2) age older than 60 years; or (3) very high platelet count (>1,500×10 9 /l). Intermediate-risk ET patients are classified as those without high-risk criteria, but with hereditary thrombophilia or cardiovascular risk factors. Low-risk ET patients are without any of these risk factors.

For high-risk patients, hydroxycarbamide is standard therapy. However, due to the potential leukemogenicity of this drug, anagrelide or pegylated-interferon-α are possible alternative options in 'young' patients. This is a difficult issue and under much debate as currently no consensus has been reached upon evaluation of the available data. One has to bear in mind that ET patients have a near-normal life expectancy, which means that they may be subject to platelet-reducing therapy for many years. Prospective studies are needed to decide whether lowering of white blood cells is an important goal of treatment in ET, allowing risk/benefit decisions in the choice of treatment in younger patients. A watch-and-wait approach is recommended for low-risk patients, though low-dose aspirin may form part of the treatment strategy in this group. Aspirin should be avoided in patients with platelet counts exceeding 1,000×10 9 /l and the combination of aspirin and anagrelide should be avoided in patients with previous major bleeding.

The platelet treatment goal is under debate. Previously, a standard goal was to lower platelet levels below 600×10 9 /l. However, this threshold appears to be based on the previous diagnostic platelet level of >600×10 9 /l, rather than evidence from clinical studies. Although there is only sporadic evidence that a lower treatment goal would provide better protection against thrombosis, since the new WHO diagnostic criteria use a platelet cut-off level of 450×10 9 /l, in the current discussion a more stringent treatment goal of 400 or 450×10 9 /l in ET patients has been suggested.

Special treatment problems

Patients may be partly unresponsive to any of the three common drugs for treating ET, or side effects may hinder a dose increase to an effective level. In these cases, combining two drugs may be an option. There are no studies exploring Fig. 1 Proposed treatment algorithm for patients with essential thrombocythaemia. Reproduced from Birgegard G. European Journal of Haematology 2007; 79 (Suppl. 68): 27-31 with permission of Blackwell Publishing. CV, cardiovascular; ET, essential thrombocythaemia; IFN, interferon-α. The age limit 60 years for the choice of firstline therapy is the personal opinion of the author such combinations, nor is any combination therapy licensed for the treatment of ET, but in clinical practice it appears to be of good use. For example, a patient develops anaemia or leukopenia from hydroxycarbamide may also benefit from receiving low-dose anagrelide, thus allowing the dose of hydroxycarbamide to be reduced and haemoglobin levels to be restored. Likewise, a combination of drugs may be beneficial for patients who cannot tolerate a dose increase of anagrelide or interferon-α to an effective level.

Patients intolerant to hydroxycarbamide, who either have cardiac insufficiency or are very old, constitute a special treatment problem. Anagrelide is not suitable in these patients, and neither is interferon-α. In this situation, busulphan may be an option, in spite of its leukaemogenic properties. A course of busulphan may provide sustained remission over many months, and the likelihood of developing leukaemia before death from other causes is low. On the same grounds, radioactive phosphorous is also an option.

During pregnancy it is not unusual for platelet levels to fall, but if platelet-reducing therapy is needed, interferon-α is the only recommended drug [111].

Possible future therapy developments

MPDs are clonal diseases and the ultimate goal must be to find a cure. However, the treatment of MPDs is a developing field. The detection of the JAK2V617F mutation may offer the opportunity for drug development and also provide a molecular marker for treatment efficacy. In PV, the reduction in JAK2 allele burden with interferon-α treatment is of considerable interest [112], and similar findings have recently been found during hydroxycarbamide therapy in PV (personal communication). However, there have been no reports as to whether this is also true for ET. It is most likely that the JAK2V617F mutation is not the only molecular event responsible for development of PV and, therefore, although JAK2 inhibitors are of great interest, they are unlikely to provide a cure. This is especially relevant in ET, where only half of patients carry the mutation, almost exclusively in heterozygous form [START_REF] Campbell | Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study[END_REF]. However, if JAK2 inhibitors prove to be effective for reducing platelet numbers, the discussion will concern whether the expected high cost of such drugs is warranted for symptomatic treatment. One can, therefore, expect the current platelet-reducing agents to maintain their place in the therapy of ET for the foreseeable future.

Conclusions

The objective of ET treatment is to reduce the risk of thromboembolic and haemorrhagic complications associat-ed with the disorder. Treatment is given according to risk stratification: platelet reduction should be given in high-risk patients and selected intermediate-risk patients, whereas no treatment or aspirin should be used in low-risk patients. Hydroxycarbamide is currently first-line therapy in patients older than 60 years of age, whereas non-mutagenic drugs like anagrelide and interferon-α might be considered in younger patients and those clinically intolerant or resistant to hydroxycarbamide.

Table 1

 1 New World Health Organization (WHO) proposed classification for essential thrombocythaemia[12]

	All the following criteria must be met to diagnose essential
	thrombocythaemia:
	& Sustained platelet count ≥450×10 9 /l (reduced from ≥600×10 9 /l)
	& Bone marrow biopsy specimen showing proliferation mainly of the
	megakaryocytic lineage with increased numbers of enlarged,
	mature megakaryocytes; no significant increase or left-shift of
	neutrophil granulopoiesis or erythropoiesis
	& Does not meet the WHO criteria for polycythaemia vera, primary
	myelofibrosis, chronic myelogenous leukaemia, myelodysplastic
	syndromes or other myeloid neoplasms
	& Demonstration of JAK2V617F mutation or other clonal marker, or in
	the absence of a clonal marker, no evidence for reactive
	thrombocytosis

Table 2

 2 Consensus definition of clinical resistance or intolerance to hydroxycarbamide in patients with essential thrombocythaemia (adapted from Barosi et al.[START_REF] Barosi | A unified definition of clinical resistance/ intolerance to hydroxyurea in essential thrombocythemia: results of a consensus process by an international working group[END_REF])

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Acknowledgements Medical writing assistance was provided by Fishawack Communications and financial support was provided by Shire.

This manuscript was supported by an unrestricted educational grant from Shire.

Conflicts of interest Prof. Dr Birgegård has received unrestricted research support from Schering-Plough and Shire, as well as fees for speaking assignments from Shire.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


E Schmitt

A D Leo

M Ho

Hensel

M Kornacker
email: martin.kornacker@arcormail.de

B Kornacker

C Schmitt

E Leo

A D Ho

M Hensel

: E Leo

Commercial LightCycler®-based quantitative real-time PCR compared to nested PCR for monitoring of Bcl-2/IgH rearrangement in patients with follicular lymphoma

Keywords: t(14;18), Quantitative PCR, Nested PCR, Follicular lymphoma, Molecular monitoring, Minimal residual disease

    Commercial LightCycler®-based quantitative real-time PCR compared to nested PCR for monitoring of Bcl-2/IgH rearrangement in patients with follicular lymphoma

Introduction

The translocation (t) (14;18) (q32;q21), juxtaposing the B cell lymphoma 2 (Bcl-2) oncogene to the immunoglobulin heavy chain (IgH) locus is a common genetic event in follicular lymphoma (FL). It can be detected in 70% of FL cases by polymerase chain reaction (PCR) spanning the major breakpoint region (mbr) of this translocation. The amplification of t(14;18) by PCR has been employed as a marker for minimal residual disease (MRD) [START_REF] Lee | Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification[END_REF] and is more sensitive than morphology, flow cytometry, or Southern blotting [START_REF] Gribben | Detection of minimal residual disease in patients with lymphoma using the polymerase chain reaction[END_REF]. It could be demonstrated that high-dose therapy [START_REF] Gribben | Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma[END_REF] as well as conventionally dosed treatment [START_REF] Lopez-Guillermo | The clinical significance of molecular response in indolent follicular lymphomas[END_REF][START_REF] Lopez-Guillermo | Molecular response assessed by PCR is the most important factor predicting failurefree survival in indolent follicular lymphoma: update of the MDACC series[END_REF] was able to achieve complete molecular responses in a significant number of patients. In these as well as in other reports [START_REF] Corradini | Long-term follow-up of indolent lymphoma patients treated with high-dose sequential chemotherapy and autografting: evidence that durable molecular and clinical remission frequently can be attained only in follicular subtypes[END_REF][START_REF] Freedman | Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma[END_REF][START_REF] Rambaldi | Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma[END_REF][START_REF] Von Neuhoff | Comparison of different strategies of molecular genetic monitoring following autologous stem cell transplantation in patients with follicular lymphoma[END_REF], MRD negativity after treatment correlates with prolonged overall and progression-free survival. Usually, semi-nested or nested PCR approaches have been used to detect t(14;18) [START_REF] Lee | Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification[END_REF]. In nested PCR, two sets of primers are used in two successive runs. The second primer set is intended to amplify a secondary target within the product of the first run. This prevents the amplification of sequences resulting from unexpected primer binding sites, thereby reducing contaminations in the PCR product and increasing sensitivity. Nested PCR is an end-point PCR, relying on the detection of the PCR product at the end of the run. In contrast, LightCycler® PCR is a real-time PCR which allows for the quantification of the amplified DNA during the linear-log phase of PCR. The PCR product is detected by fluorescence using two short oligonucleotides which are each labelled with a different fluorescent dye. They hybridise to an internal sequence of the amplified fragment. After hybridisation to the template DNA, the two probes come in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorescent dyes. FRET causes them to emit fluorescence at a specific wavelength which correlates to the amount of PCR product and is measured by the LightCycler® apparatus. Results are expressed as the target/reference ratio of each sample normalised by the target/reference ratio of a standardised DNA sample (calibrator). Recently, a LightCycler®based commercial assay to quantify cells harbouring t(14;18) has become available. The target concentration in each sample is calculated relative to the housekeeping gene tissue plasminogen activator (tPA). In this paper, we report on the comparison of a nested PCR assay with the LightCycler® t(14;18) quantification kit (mbr; Roche Diagnostics) in a homogeneous population of patients with FL positive for t(14;18).

Materials and methods

Patients and patient samples

Samples analysed in this study represent a subpopulation of patients enrolled in the randomised phase II multicenter study HD2000 on whom both nested PCR as well as quantitative real-time LightCycler® PCR were performed and at least one of the samples collected was positive for t(14;18) by nested PCR. Thus, all patients reported here were considered to bear t(14;18) within their lymphoma cells. Blood and bone marrow samples were collected prospectively from follicular lymphoma patients treated within this trial with CHOP chemotherapy and rituximab (Mabthera, Roche, Grenzach-Wyhlen, Germany). The aim of the clinical study was to determine the influence of combined administration of CHOP chemotherapy with varying numbers of rituximab infusions as first-line treatment on the remission quality/duration of patients with FL. All patients investigated in this study have given written informed consent before entering the study. The study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki. To be eligible, patients were required to have proven diagnosis of CD20 positive FL (grades I, II and III) according to the REAL classification and to be older than 18 years of age with clinical stages III-IV (according to Ann Arbor staging system). Characteristics of patients are summarised in Table 1. Patients received 6 cycles of CHOP (cyclophosphamide 750 mg/m 2 , doxorubicin 50 mg/m 2 , vincristine 1.4 mg/m 2 and prednisone 100 mg) chemotherapy every 21 days. Furthermore, patients were randomly assigned to receive rituximab (375 mg/m 2 ) administered by one, three or six intravenous infusions either with the first, the first to third or the first to sixth cycle of CHOP, respectively. Rituximab was always administered 1 day prior to chemotherapy. Samples were taken before therapy and after 3 and 6 cycles of therapy. Overall, 249 PCRs were performed on samples from blood and from bone marrow collected from 28 patients. Fifty-six bone marrow samples were analysed with a nested PCR and 54 with a quantitative real-time LightCycler® PCR. Seventy-five samples derived from the peripheral blood of the patients were analysed with the nested and 64 with the LightCycler® PCR.

Polymerase chain reaction

Isolation of DNA

Samples from peripheral blood and bone marrow aspirations were taken before therapy and after three and six treatment cycles. Total cellular DNA was extracted using the QIAamp DNA blood maxi kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.

For quantitative analysis, a real-time detection of t(14;18) using the commercially available "LightCycler® t(14;18) quantification kit (mbr)" (Roche Diagnostics) was performed according to the manufacturer's recommendations. Final reaction volume was 20 μl containing 5 μl of DNA and 15 μl of the reaction mix which includes FastStart Taq DNA polymerase, Mg ++ , a forward primer binding to exon 3 of mbr, a reverse primer binding to JH segment and fluorescently labelled hybridisation probes. The amount of DNA added to the reaction ranged from 100 to 7180 ng (mean 1,290 ng, SD±1,054 ng). The mixture was transferred into Results are quantified with the LightCycler® relative quantification software (Roche Diagnostics), and the final results are expressed as the ratio of target copies [t(14;18)] to reference copies (tPA) in the sample relative to the ratio of target to reference copies in the calibrator. Thus, a value of 1 would indicate that each cell of the sample contained t (14;18), and a value of, e.g. 10 -3 would indicate that one cell in 1,000 cells would contain the translocation, i.e. one t (14;18) positive follicular lymphoma cell would be found within 1,000 normal blood or bone marrow cells.

For nested PCR, the mbr of the t(14;18) translocation was amplified by a two-step nested PCR. The first PCR reaction was performed with 500 ng of DNA in a final volume of 50 μl using primers Bcl-2NFO (5′-GAC CAG CAG ATT CAA ATC TAT GGT GGT-3′) and JHNRO (5′-GGA CTC ACC TGA GGA GAC GGT GA-3′) in a concentration of 0.36 μM each, 54 °C annealing temperature and 25 cycles. Three microlitres of the PCR product was subjected to the second PCR reaction containing the inner primers Bcl-2NFI (5′-CCT TTA GAGAGT TGCTTTACGTGGCC-3′) and JHNRI (5′-GGAGACGGT GAC CAG GGT-3′) in a concentration of 0.36 μM each. The second PCR reaction was performed using 55°C annealing temperature and repetitions of 30 cycles. Samples were tested as duplicates in three independent runs. They were scored as PCR-positive when at least one of six reactions showed a specific amplification product. Nested PCR was shown to detect one positive cell in 10 5 normal cells [START_REF] Schmitt | The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity[END_REF].

Statistics

Values of p to compare the percentage of positive results between different sample groups were calculated using Fisher's exact t test. Differences in LightCycler® PCR results were measured using the Wilcoxon two-sample test.

McNemar's test was used to compare nested PCR and LightCycler® PCR results for Bcl-2/JH rearrangement detection on the same samples.

Results

Serial tenfold dilutions of the t(14;18) harbouring cell line K422 in peripheral blood mononuclear cells from normal donors established a sensitivity of the LightCycler® PCR assay between one t(14;18) positive cell in 10 5 -10 4 normal cells and a linear measuring range at dilutions of K422 cells between 10 0 and 10 -4 . These results are similar to data provided by the manufacturer and to data described before [START_REF] Martin | LightCycler-based quantitative real-time PCR monitoring of patients with follicular lymphoma receiving rituximab in combination with conventional or high-dose cytotoxic chemotherapy[END_REF] and are therefore not shown.

Sensitivity of quantitative vs. nested PCR

To assess sensitivity of nested and LightCycler® PCR, all 117 samples on which both PCR methods had been performed were evaluated regarding PCR status and displayed in a 2×2 contingency table (Table 2). The bcl2-IgH rearrangement was detectable in 42% of samples by nested but not by LightCycler PCR. Samples with positive results in LightCycler PCR were always positive with nested PCR (p<0.0001, Table 2).

Molecular disease status before and after therapy

To further compare nested PCR with LightCycler® PCR, results from bone marrow and peripheral blood samples taken before start of therapy and after 3 and 6 cycles of immunochemotherapy were analysed (Figs. 1 and2). A summary of results can be found as Electronic Supplementary Material. With nested PCR, 23 of 25 (93%) bone marrow samples were positive before therapy, four of 13 (31%) after 3 cycles and four of 18 (22%) after 6 cycles of chemotherapy. With peripheral blood samples, 25 of 28 (89%) were positive before therapy, five of 21 (24%) after 3 cycles and five of 23 (22%) after 6 cycles of therapy. Differences between samples obtained before therapy and after 3 or 6 cycles were highly significant (Fig. 1).

With LightCycler® PCR for bone marrow samples, the mean relative t(14;18)/tPA ratio before therapy (4.1×10 -3 ) dropped to 2.17×10 -5 after 3 cycles and to 8.12×10 -6 after 6 cycles of therapy. In peripheral blood, the mean t(14;18)/ tPA ratio before therapy was lower (4.08×10 -4 ) than in bone marrow samples and dropped to 1.59×10 -6 after 3 cycles and to 7.89×10 -6 after 6 cycles of therapy (Fig. 2). In bone marrow as well as in peripheral blood, only few samples remained positive after therapy, but the observed reduction in lymphoma cells during treatment was only significant for bone marrow when results before were compared to results after 6 cycles of therapy.

Molecular disease status dependent on bone marrow involvement

To correlate PCR results to histomorphological measures of tumour involvement, we performed nested as well as LightCycler® PCR on bone marrow and peripheral blood samples from patients with or without histological proven BM infiltration. With nested PCR, the majority of samples from BM as well as from PB proved to be positive whether bone marrow was infiltrated by lymphoma cells or not (Fig. 3). In BM, 100% of samples were positive in cases with and 87% were positive in samples without lymphoma involvement. Similarly, 92% of PB samples were positive when BM was infiltrated by FL and 87% were positive in cases without BM infiltration. With LightCycler® PCR, the mean t(14;18)/tPA ratio was higher in the samples from patients with histological proven BM infiltration of lym- Fig. 1 Nested PCR for t(14;18) in bone marrow and peripheral blood samples of FL patients taken before start of therapy and after 3 and 6 cycles of immunochemotherapy. Twenty-three of 25 (93%) of bone marrow samples were positive before therapy, four of 13 (31%) after 3 cycles and four of 18 (22%) after 6 cycles of chemotherapy. With peripheral blood samples, 25 of 29 (86%) were positive before therapy, five of 22 (23%) after 3 cycles and five of 24 (21%) after 6 cycles of therapy Fig. 2 LightCycler® PCR for t(14;18) in bone marrow and peripheral blood samples of FL patients taken before start of therapy and after 3 and 6 cycles of immunochemotherapy. The mean relative t(14;18)/tPA ratio before therapy (4.1×10 -3 ) dropped to 2.17×10 -5 after 3 cycles and 8.12×10 -6 after 6 cycles of therapy. In peripheral blood, the mean t(14;18)/tPA ratio before therapy was lower (4.08×10 -4 ) than in bone marrow samples and dropped to 1.59×10 -6 after 3 cycles and to 7.89×10 -6 after 6 cycles of therapy Fig. 3 Nested PCR for t(14;18) on bone marrow and peripheral blood samples from patients with or without histological proven BM infiltration by FL. With nested PCR, the majority of samples from BM as well as from PB proved to be positive whether bone marrow was histologically infiltrated by lymphoma cells or not phoma cells (1.9×10 -2 ) compared to samples from patients without BM involvement (4.08×10 -5 , Fig. 4). A similar trend was seen in samples derived from the peripheral blood, albeit at lower overall values (1.03×10 -3 with, 2.1× 10 -5 without BM involvement). These correlations were not statistically significant.

Correlation between MRD after therapy and relapse

We were interested if PCR results at the end of therapy would be able to predict if these patients will suffer from early relapse. We were able to obtain PCR results after 6 cycles of therapy as well as information on the remission status 6 months later on 13 patients. Eleven of these stayed in remission (CR=6, PR=4, SD=1), whereas two suffered from PD. The two patients with PD had achieved a PR after therapy, and with nested PCR, they had a positive result in their BM as well as in their PB samples at that time (Fig. 4).

Of the 11 patients remaining in remission, one was positive in BM and two in PB with nested PCR. With LightCycler® PCR, the two patients with PD had divergent results in BM and PB after therapy. None of the 11 patients in remission had a positive LightCycler® result at the end of therapy.

The PCR results before, during and after therapy of the two relapsing patients are shown in Table 3.

Discussion

We have compared nested and quantitative LightCycler®based PCR for detection of t(14;18) in patients with FL. Sensitivity was one t(14;18) positive cell in 10 5 negative cells for nested and one in 10 5 to one in 10 4 for LightCycler® PCR. This difference resulted in a significant larger number of t(14;18)-positive blood and bone marrow samples obtained before therapy with nested compared to LightCycler® PCR. A likely explanation is that at this time point, a significant proportion of patients harbour between 1×10 -4 and 1×10 -5 FL cells in their circulation and in their bone marrow, and these positive cases are missed when the less sensitive LightCycler® PCR method is utilised. A lesser sensitivity of real-time vs. nested PCR has also been reported in patients with chronic myeloid leukemia. With real-time PCR, 12 of 94 bone marrow samples were negative for the disease marker BCR-ABL. With nested RT-PCR, in six of these 12 patients, BCR-ABL transcripts could be detected after the second round of amplification [START_REF] Schoch | Comparison of chromosome banding analysis, interphase-and hypermetaphase-FISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic myeloid leukemia: a study on 350 cases[END_REF].

Fig. 4 LightCycler® PCR for t(14;18) on bone marrow and peripheral blood samples from patients with or without histologically proven BM infiltration by FL. With LightCycler® PCR, the mean t(14;18)/tPA ratio was higher in the samples from patients with histologically proven BM infiltration of lymphoma cells (1.9×10 -2 ) compared to samples from patients without BM involvement (4.08×10 -5 ). A similar trend was seen in samples derived from the peripheral blood, albeit at lower overall values (1.03×10 -3 with, 2.1×10 -5 without BM involvement) A hint for the clinical relevance of PCR testing would be if a therapeutic intervention is reflected by PCR results. Indeed, this is the case for nested PCR: Three or 6 cycles of immunochemotherapy resulted in a significant drop in PCR-positive samples in blood and in bone marrow, demonstrating that t(14;18)-positive cells drop below 1× 10 -5 in these patients after therapy. LightCycler®-based PCR also showed a reduction of t(14,18) bearing cells and an increase in negative samples, but without reaching statistical significance. This may have been due to the low sensitivity of the assay, with 14 of 24 and 17 of 26 pretherapy samples being negative with LightCycler® PCR in bone marrow and peripheral blood, respectively.

Similar to the study reported here, Martin et al. [START_REF] Martin | LightCycler-based quantitative real-time PCR monitoring of patients with follicular lymphoma receiving rituximab in combination with conventional or high-dose cytotoxic chemotherapy[END_REF] have used the commercially available LightCycler® t(14;18) quantification kit (mbr) in patients with follicular lymphoma. Contrary to our finding, they were able to describe a significant correlation between LightCycler® PCR results and lymphoma activity [t(14;18)-positive cells in BM or PB before vs. after therapy]. Several factors may be responsible for this discrepancy:

-We did observe a higher number of samples being negative at initial diagnosis (58% in blood and 65% in bone marrow samples) compared to the samples analysed by Martin et al. (46% and 52%, respectively) with LightCycler PCR. Correspondingly, median t(14;18)/tPA ratio was lower at initial diagnosis with zero as the median in blood and bone marrow samples in our patients compared to a median of 5×10 -5 and 6×10 -5 , respectively, in the series presented by Martin et al.. This may have been caused by differences in sample handling and DNA isolation. Even when similar protocols are used, quantitative results may vary: A two log variation in the number of positive cells has been reported in an international study on 12 laboratories performing quantitative PCR for t(14;18) on aliquots of blood spiked with defined numbers of lymphoma cells [START_REF] Darby | Variability of quantitative polymerase chain reaction detection of the bcl-2-IgH translocation in an international multicenter study[END_REF].

-Almost one quarter of the patients analysed by Martin et al. had relapsed FL, which may have resulted in more aggressive disease with more lymphoma cells disseminating into PB or BM compartments. -Patient numbers were smaller in our study. t(14;18) is not specific for malignant disease. This translocation has been detected in lymph nodes and tonsils with benign follicular hyperplasia [START_REF] Limpens | Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia[END_REF] and in blood and spleen cells of healthy volunteers [START_REF] Schmitt | The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity[END_REF][START_REF] Limpens | Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals[END_REF][START_REF] Liu | BCL2 translocation frequency rises with age in humans[END_REF][START_REF] Summers | Frequency of the Bcl-2/IgH rearrangement in[END_REF]. We have reported previously that 24% of healthy blood donors carried the translocation when analysed with nested PCR and 19% were positive with a real-time quantitative PCR. In that study, detection limit for both assays was one t(14;18) copy in 10 5 cells [START_REF] Schmitt | The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity[END_REF]. Thus, we cannot exclude that part of the positive results reported here are derived from t(14;18) in non-malignant lymphocytes unrelated to follicular lymphoma. The majority of healthy individuals being positive for t(14;18) carry the translocation at a frequency below 10 -4 [18]. In another study with 25% of healthy volunteers being positive for t(14;18), only 3% were positive at frequencies of more than one in 10 4 cells [START_REF] Summers | Frequency of the Bcl-2/IgH rearrangement in[END_REF]. A clear correlation between PCR sensitivity and the prevalence of circulating t(14;18)-positive cells has been demonstrated in a meta-analysis of 13 studies with 1,908 healthy individuals [19]. These findings suggest that nested PCR will detect t(14;18) unrelated to follicular lymphoma more often than LightCycler® PCR, resulting in false positive results. A comparison of DNA sequences between PCR products and the initial lymphoma sample will be important to elucidate the origin of the translocated genes in future studies.

Nested PCR was not able to differentiate between bone marrow samples with or without histological proven involvement: All samples with lymphoma involvement and 87% of samples without involvement were t(14;18)positive. In contrast, LightCycler® PCR showed lower number of cells with t(14;18) and a higher percentage of negative samples in bone marrow not infiltrated with follicular lymphoma, albeit missing statistical significance. This does suggest that the number of t(14;18)-positive cells is above 10 -5 in the majority of BM samples independent of lymphoma infiltration, preventing nested PCR to differentiate between samples with or without histological evidence of involvement.

Interestingly, with LightCycler PCR, there was also a correlation between BM involvement and t(14;18) cell numbers in the peripheral blood. This could point towards a spill-out phenomenon, allowing FL cells to enter circulation from the BM compartment. On the other hand, this correlation could merely reflect increased tumour load or biologically more aggressive disease in patients with BM involvement, leading to higher numbers of circulating lymphoma cells. A correlation between histological marrow infiltration and higher levels of BLC2/JH fusion sequences with real-time PCR in BM [20] and PB [20,21] has been demonstrated before.

The data shown here demonstrate a lower sensitivity of LightCycler® vs. nested PCR for detection of t(14;18). This has implications for the selection of the most suitable PCR method when analysing clinical samples: In samples where the ratio of target to reference gene is approaching the limit of detection of LightCycler® PCR, e.g. after immunochemotherapy, only nested PCR may be able to demonstrate the effect of a therapeutic intervention on residual lymphoma cells. On the other hand, with numbers of positive cells well above the limit of detection in the majority of samples, as in the case of stage IV disease before therapy, LightCycler® PCR may be well able to quantitate t(14;18) in clinical samples.

Most, if not all, patients will ultimately relapse after immunochemotherapy for follicular lymphoma. Consolidation or maintenance therapy, e.g. with rituximab, may help to prevent or delay clinical relapses [22][23][24][25]. If it would be possible to predict disease control after first-line therapy, these therapies could be selected for patients with a high likelihood of early relapse. With qualitative PCR, several studies were able to document a relationship between molecular response and time to relapse in patients treated with standard-dose chemotherapy [START_REF] Lopez-Guillermo | The clinical significance of molecular response in indolent follicular lymphomas[END_REF][START_REF] Lopez-Guillermo | Molecular response assessed by PCR is the most important factor predicting failurefree survival in indolent follicular lymphoma: update of the MDACC series[END_REF]26], with high-dose chemotherapy [START_REF] Freedman | Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma[END_REF][27][28][29][30] and with rituximab-containing regimen [START_REF] Rambaldi | Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma[END_REF]31,32]. Due to the low number of patients and the limited follow-up time of 6 months, we can only speculate on the role of molecular diagnostics for the prediction of relapsed disease. The two patients in our study relapsing within 6 months after therapy were positive in BM and PB with nested PCR at the end of therapy and could have been chosen to receive maintenance treatment. At the same time, positive results in patients who did not relapse occurred, albeit at low rates. With LightCycler® PCR, BM or PB samples of the two relapsing patients were not invariably positive at the end of therapy.

Quantitative PCR may offer additional benefits as it may (a) allow for the identification of patients with an increase in MRD after therapy, being at an increased risk of relapse [33][34][35] and (b) stratify patients according to the initial tumour load in BM, thereby predicting response to therapy and long-term outcome [36]. Our results suggest that the relatively low sensitivity of the LigthCycler assay may be disadvantageous for the prediction of early relapse after therapy. This may be particularly relevant for rituximabcontaining regimen where a single antibody infusion added to 6 cycles of CHOP chemotherapy resulted in MRD negativity in the majority of patients [37]. In contrast, nested PCR for the bcl2-IgH rearrangement might be better suited for predicting early relapse after primary therapy. Its use for decision making regarding maintenance therapy has to be validated in larger trials.

Table 1

 1 Patients' characteristics

	Patient number	28
	Age (median and range)	54 (34-69)
	Sex (m/f)	(16/12)
	Clinical stage
	III	11
	IV	17
	Histological grade
	I	1 2
	II	9
	III	3
	No data	4

Table 2

 2 Negative and positive results with nested and LightCycler® PCR: 2×2 contingency table summarizing results on all 117 BM and PB samples of which nested as well as quant. LightCycler® PCR results were available

	Nested PCR	Total
	Negative	Positive

Table 3

 3 Time course of nested and LightCycler® PCR results in the two patients having relapsed 6 months after therapy

		Before	After 3 cycles	After 6 cycles	6 months after therapy
	Patient A
	Remission status	NA	PR	PR	PD
	Nested PCR-BM	Positive	Positive	Positive
	LightCycler® PCR-BM	6.12×10 -2	2.6×10 -4	1.38×10 -4
	Nested PCR-PB	Positive	Positive	Positive
	LightCycler® PCR-PB	ND	Negative	Negative
	Patient B
	Remission status	NA	PR	PR	PD
	Nested PCR-BM	Positive	Positive	Positive
	LightCycler® PCR-BM	1.39×10 -5	Negative	Negative
	Nested PCR-PB	Positive	Positive	Positive
	LightCycler® PCR-PB	5.49×10 -5	ND	1.5×10 -4

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Acknowledgment This work was supported in part by a grant of the Medical Faculty of the University of Heidelberg.