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http://msj.sagepub.com/content/16/12/1490The online version of this article can be found at:

 DOI: 10.1177/1352458510379613

2010 16: 1490 originally published online 1 September 2010Mult SclerAssunta Rocca, Mariaemma Rodegher and Giancarlo Comi

Federica Esposito, Marta Radaelli, Vittorio Martinelli, Maria Pia Sormani, Filippo Martinelli Boneschi, Lucia Moiola, Mariaprogressive multiple sclerosis

remitting and secondary−−Comparative study of mitoxantrone efficacy profile in patients with relapsing  

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Research Paper

Multiple Sclerosis

16(12) 1490–1499

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DOI: 10.1177/1352458510379613

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Comparative study of mitoxantroneefficacy profile in patients withrelapsing–remitting and secondaryprogressive multiple sclerosis

Federica Esposito1,2,*, Marta Radaelli1,2,*, Vittorio Martinelli1,2,Maria Pia Sormani3, Filippo Martinelli Boneschi1,2,Lucia Moiola1,2, Maria Assunta Rocca2,4,Mariaemma Rodegher1,2 and Giancarlo Comi1,2

Abstract

Background: Mitoxantrone (MTX) is an immunosuppressive drug approved for multiple sclerosis (MS) treatment.

Objective: The aim of this study is to evaluate and to compare the clinical and neuroradiological responses to MTX in

relapsing–remitting (RR) and secondary progressive (SP) MS patients.

Methods: We conducted a retrospective, non-randomized, open-label, observational study to evaluate the clinical and

neuroradiological response to the drug in 79 patients with RR MS and 210 patients with SP MS.

Results: A statistically significant reduction (p< 0.001) in the number of relapses was observed during MTX treatment

and in the year after in both RR and SP MS patients. On the contrary, an opposite behavior in terms of disease

progression was found in RR compared with SP MS patients, resulting in a statistically significant improvement of the

Expanded Disability Status Scale score during the MTX treatment (p< 0.001) and in the year after (p< 0.001) for RR MS

patients compared with a continuous, although mild, worsening of the disability in SP MS patients (p< 0.001). Finally, a

significant reduction (p< 0.001) of new Gd-enhanced lesions in both RR and SP MS patients was observed in a subgroup

of 224 individuals who underwent a brain MRI evaluation before and after MTX treatment.

Conclusions: MTX should be considered as an effective therapeutic option in RR MS patients with evidence of relevant

disease activity, but the potential life-threatening adverse events and the overall benefit–risk ratio must be carefully

evaluated at individual patient level.

Keywords

multiple sclerosis, secondary progressive, relapsing–remitting

Date received: 1st March 2010; revised: 21st June 2010; accepted: 22nd June 2010

Introduction

Multiple sclerosis (MS) is an autoimmune, T-cell-mediated demyelinating disorder of the central nervoussystem (CNS), characterized by the combination ofinflammation and neurodegeneration. All of the cur-rent available MS treatments target the inflammatoryphase, and can be generally classified as immunomodu-lants (interferons beta-1a and beta-1b, glatirameracetate), immunosuppressants (mitoxantrone, cyclo-phosphamide) or monoclonal antibodies (natali-zumab). Mitoxantrone (MTX) is the first drugapproved in 2000 by the Food and DrugAdministration (FDA) for the treatment of active ‘pro-gressive’ MS, under the following indication: ‘for

1Department of Neurology, Division of Neuroscience, Scientific Institute

San Raffaele, Milan, Italy.2Institute of Experimental Neurology, Scientific Institute San Raffaele,

Milan, Italy.3Biostatistics Unit, Department of Health Sciences (DISSAL), University

of Genoa, Genoa, Italy.4Neuroimaging Research Unit, Scientific Institute San Raffaele, Milan, Italy.

*These authors contributed equally to this work.

Corresponding author:

Federica Esposito, MD, Department of Neurology, Scientific Institute San

Raffaele, Via Olgettina 48, 20132 Milan, Italy

Email: esposito.federica@hsr.it

reducing neurological disability and/or the frequency ofclinical relapses in patients with secondary progressive(SP), progressive–relapsing (PR) or worsening relaps-ing–remitting multiple sclerosis (WRR MS)’.

MTX is an anti-neoplastic anthracenedione deriva-tive which inhibits DNA replication, DNA-dependentRNA synthesis and DNA repair. The mechanisms ofaction of MTX in MS include also: (1) a selective downregulation of CD4 cells,1 resulting in a strong lympho-cytopenia; (2) suppressive effects on different B cellfunctions, such as antigen presentation, antibody-dependent demyelination and complement-mediatedmyelinolysis;2–4 (3) an inhibition of the demyelinatingactivity of macrophages; and (4) a proliferation of non-specific suppressor T cells.1 MTX is a very small mol-ecule which is able to cross the blood–brain barrier andto interact with cells in the CNS; it is characterized by alow oral absorption and by a preferred intravenousroute of administration. MTX is a long-acting immu-nosuppressor; it is rapidly absorbed by tissues and thensequestered in the deep tissue compartment for anextended period of time (up to 1 month).5

The first observations supporting MTX efficacy inMS came from studies performed in experimental aller-gic encephalomyelitis (EAE); specifically, a suppressionof EAE evolution was observed after the intravenousadministration of the drug.6 Phase II and phase IIIclinical trials7–10 provided further evidences of MTXefficacy in reducing relapse-rate and disability progres-sion in RR and SP MS. In line with that, a Cochranemeta-analysis confirmed the short-term efficacy of thedrug in MS patients.11

The most important limitation in the drug use isrepresented by its long-term safety, mainly related tothe risk of cardiotoxicity and acute leukemia (AL).12 Acontroversial debate on the actual AL risk is ongoing inthe scientific community across different countries.As regards the risk of cardiotoxicity, it is knownthat it increases with a cumulative dose greater than100mg/m2,13 leading to the recommendation of notexceeding a maximum lifetime dose of 140mg/m2.14

In March 2005, a warning from the FDA recommendedalso to perform a periodic monitoring of the cardiacfunction before each MTX dose. Other side-effects arerepresented by the risk of amenorrhea, which can alsobe persistent, reduction of fertility and severeinfections.

Studies on MS pathophysiology and pathogenesissuggest that the mechanism responsible for damagemay be at least partially different in the RR and SPphases of the disease, with prevalent T-cell-mediateddamage in the first disease course and prevalent micro-glia and antibody-mediated damage in the second typeof disease.15 Moreover, the SP phase seems to be char-acterized by a compartmentalization of the CNS

inflammation, which is supposed to be trapped behinda closed or repaired blood–brain barrier. Such differ-ences are probably responsible for the differentresponses to immunomodulant and immunosuppres-sive treatments in RR and SP MS patients.

The aim of this observational study is to evaluateand to compare the clinical and neuroradiologicalresponses to MTX infusion in a large group of RRand SP MS patients, in order to better define the ben-efit–risk ratio of the treatment in patients with differentdisease courses.

Patients and methods

Patients affected by RR or SP MS (according to theMcDonald criteria16,17), who referred to theDepartment of Neurology at the Scientific InstituteSan Raffaele in Milan, were enrolled in this study. Inorder to be included, patients had to have an aggressivedisease or no adequate response to ongoing immuno-modulatory treatments, defined on an ExpandedDisability Status Scale score worsening of at least onepoint (unconfirmed or confirmed at 3 months) in the2 years before the enrolment in either disease category.In addition, they had to have evidence of an active dis-ease: at least one attack in the previous 6 months or atleast one Gd-enhanced lesion in a brain MRI performedin the previous 6 months. Only patients who received atleast five cycles of MTX were considered in this study, inorder to exclude subjects who received ‘short induction’treatment schemes. Patients with cardiac left-ventricularejection fraction (LVEF) <50%, hepatic, renal or pul-monary disease, pregnant or breast-feeding women orsubjects with any other condition which precludedMTX treatment were also excluded.

Clinical evaluation was performed at baseline andevery 3–6 months in the 2 years before treatment.During the MTX treatment period and in the yearafter the end of MTX, the patients were evaluatedapproximately every 3 months. Additional visits wereplanned when required. Clinical assessment included athorough neurological examination with evaluation bythe Expanded Disability Status Scale.18 In a subgroupof patients, a brain MRI performed within a 3-monthwindow before and after MTX treatment was available.For each scan, the presence of Gd-enhanced lesions wasevaluated (active MRI).

On the day of infusion, the patients underwentan electrocardiography and all females of child-bearing potential performed a pregnancy test.Echocardiography was performed initially at baselineand after six cycles. After the FDA recommendationson MTX cardiotoxicity, the echocardiography was per-formed at least every three cycles. In the case of animportant reduction of LVEF (ejection fraction below

Esposito et al. 1491

50% or a difference between baseline and on-treatmentevaluation greater than 10%), echocardiography wasrepeated after 1 month and, if a persistent reductionwas confirmed, the treatment was permanently discon-tinued. All adverse events reported by the patients,either spontaneously or on questioning, were recorded.

Treatment

The treatment protocol was 10mg/m2 of MTX monthlyfor 3 months, followed by 10mg/m2 every 2 months,with a therapeutic window of �2 weeks. The drug wasintravenously administered via a slow infusion over30–60minutes. An anti-emetic drug was used beforeeach cycle, while a physiologic solution of 250ml wasgiven after the infusion. No steroid treatment was com-bined to MTX infusion on the day of administration.Hematological data were collected for all the patientsat different time points: one week before MTX infusion,during the day ofMTX infusion, between 10 and 14 daysafter MTX infusion and after 30 days from the treat-ment. MTX dosage was reduced to 5mg/m2 if one ofthe following conditions was detected at the hematolog-ical exams performed one week before the MTX infu-sion: leukopenia (white blood cell count between 3.0 and4.0�109/L, or neutrophils between 1.3 and 1.5�109/L,or lymphocytes between 0.8 and 0.9�109/L); or throm-bocytopenia (platelet count between 75 and 100�109/L).The treatment was temporarily interrupted or delayed inthe case of severe leukopenia (white blood cell countbelow 3.0�109/L, or neutrophils below 1.3�109/L, orlymphocytes below 0.8�109/L) or thrombocytopenia(platelet count below 75�109/L).

Statistical analysis

Baseline demographic and clinical characteristics of RRand SP MS patients were compared using Student’st-test.

The clinical and neuroradiological efficacy of MTXtreatment was assessed in the two disease subgroups,considering the following endpoints:

Themean ExpandedDisability Status Scale change at theend of the treatment and in the follow-up period,adjusted for the Expanded Disability Status Scalescore in the 2 years before the treatment, was analyzedusing the ANOVA model. The Expanded DisabilityStatus Scale score was confirmed after 3 months.

The proportion of progression-free patients in the2 years before MTX, during the treatment periodand after MTX was assessed using McNemar’s test.

The annualized relapse rate in the 2 years before MTX,during the treatment period and after MTX wasassessed using Wilcoxon’s test.

The proportion of patients with active MRI at baselineand at the end of the MTX treatment period wascompared by McNemar’s test.

Results

Demographic characteristics

Between January 2000 and June 2007 a total of 289patients satisfied the inclusion criteria and were enrolledin the study. Themain clinical and demographic featuresat baseline are shown in Table 1. Seventy-nine patientshad a RR course and 210 had a SP course; the two sub-groups were comparable in terms of male/female ratio,mean increase of Expanded Disability Status Scale scorein the 2 years before the beginning of MTX (1.1 in RRand 1.3 in SP MS patients) and treatment duration. Inthe 2 years preceding MTX treatment, 140 patients(48%) had received immunomodulatory therapies (gla-tiramer acetate or beta-interferons), 29 patients (10%)had been treated with oral immunosuppressive drugs(azatioprine or methotrexate), and 15 (5%) had receivedcyclophosphamide, while 29 patients (10%) had neverbeen treated.

Compared with RR MS patients, SP MS patientswere older (mean age 41.1), with a longer disease dura-tion (11.4 years), and had a more severe disability(median Expanded Disability Status Scale score 5.5).The mean relapse rate in the 2 years before MTX washigher in RR than in SP MS patients (3.1 vs. 1.4). In the2 years before MTX at least one relapse was observedin 59% of SP MS patients. The patients received onaverage seven cycles of MTX (range 5–12), with amean cumulative dose of 58mg/m2 and 62mg/m2 inRR and SP MS patients respectively. The mean treat-ment duration was 1.7 years (range 0.5–8.8), with nodifference between RR and SP MS patients.

Clinical outcomes

RR MS patients who had had a mean ExpandedDisability Status Scale score worsening from 2.0 to3.0 (p< 0.001) in the 2 years before the beginning ofMTX showed a significant improvement of disability(mean 3-month confirmed Expanded Disability StatusScale score from 3.0 to 2.6, p< 0.001) during the treat-ment. Such an improvement usually occurred in thefirst trimester after MTX beginning and then remainedstable during the 1-year follow-up period. In SP MSpatients who had had a mean increase of disabilityfrom 4.2 to 5.3 (p< 0.001) during the 2 years beforethe treatment, a slight disability worsening continuedover the course of treatment (mean 3-month confirmedExpanded Disability Status Scale score from 5.3 to 5.7,p< 0.001) and in the year after (p< 0.001).

1492 Multiple Sclerosis 16(12)

The between-group comparison of ExpandedDisability Status Scale change showed no differencebetween RR and SP MS subgroups during the 2 yearsbefore MTX treatment. Conversely, during MTX treat-ment the Expanded Disability Status Scale score signif-icantly improved in RR MS patients (p< 0.001). Afurther mild beneficial effect was also observed duringthe follow-up period (p¼ 0.01) (Figure 1). As regardsSP MS, in order to assess if the treatment of patients inthe early phase of disease worsening can positivelyaffect the response to MTX, we divided SP patientsinto two categories. Specifically, we evaluated individ-uals who received MTX within the first 6 months fromthe clinical worsening and we compared them withpatients who received the treatment after the first6 months of disease progression. The onset of the pro-gressive phase was available for 168 out of the 210 SPMS patients. We observed a similar clinical responseduring the MTX treatment between the two subgroups.However, in the 1-year follow-up there was evidence ofa less severe progression rate (p¼ 0.047) for patientstreated early in the progressive phase (n¼ 53),compared with patients treated later (n¼ 115).Similar results were obtained after comparing SP MSpatients who were treated within 12 months from thebeginning of the progressive phase with patients treatedlater.

We considered the proportion of progression-freepatients, defined as those having a 3-month confirmedExpanded Disability Status Scale increase �0.5 if thebaseline Expanded Disability Status Scale score was<6.0, and patients without any Expanded DisabilityStatus Scale increase if the baseline ExpandedDisability Status Scale score was �6.0. We observedan increased proportion of progression-free patientsduring the treatment period and in the 1-year follow-up, compared with the 2 years before MTX, in the RR

and SP MS patients (Table 2). Specifically, in the RRsubgroup, the progression-free patients were 34.2%,94.9% and 65.8% respectively in the 2 years beforethe MTX treatment, during the treatment and in the1-year follow-up (p< 0.001). In the SP subgroup, in thesame intervals of time, the proportion of progression-free patients was 27.6%, 71.0% and 65.7% respectively(p< 0.001).

We observed also an increased proportion ofrelapse-free patients in both disease subgroups duringthe treatment period (0% vs. 63% in RR MS patientsand 41% vs. 82% in SP MS patients, p< 0.001) and

Table 1. Demographic and clinical characteristics of the multiple sclerosis (MS) patients

Total MS patients(N¼ 289)

RR MS patients(N¼ 79)

SP MS patients(N¼ 210) p-value

Gender (male/female) 101/188 23/56 78/132 n.s.

Age (mean, range) 39.3 (15.4–69.5) 34.5 (15.4–58.8) 41.1 (16.6–69.5) <0.001

Disease duration (mean, range) 9.9 (0.3–47.1) 5.9 (0.3–22.2) 11.4 (1.0–47.1) <0.001

� EDSS in the 2 years before MTX* 1.2 1.1 1.3 n.s.

EDSS score at baseline (mean, range) 4.7 (1.0–8.0) 3.0 (1.0–6.0) 5.3 (3.5–8.0) <0.001

Number of relapses in the 2 years before MTX(mean, range)

1.9 (0–11) 3.1 (0–11) 1.4 (0–10) <0.001

Duration (year) of MTX treatment (mean, range) 1.7 (0.5–8.8) 1.4 (0.5–6.6) 1.8 (0.6–8.8) n.s.

Number of MTX cycles (mean, range) 7 (5–12) 7 (5–11) 7 (5–12) n.s.

MTX cumulative dose (mg/m2) (mean, range) 61 (30–100) 58 (35–90) 62 (30–100) n.s.

*Difference between Expanded Disability Status Scale score measured at the first MTX infusion and Expanded Disability Status Scale score measured at

2 years before the treatment. Disease duration and MTX treatment duration are expressed as years.

n.s., not significant; EDSS, Expanded Disability Status Scale, MTX, mitoxantrone.

p=n.s.

p<0.001 p=0.01

2 yearsbefore MTX

MTX 1-year Fw-up

ED

SS

6.5

6

5.5

5

4.5

4

3.5

3

2.5

2

1.5

RR

SP

Figure 1. Expanded Disability Status Scale changes before,

during and after mitoxantrone (MTX) treatment in relapsing–

remitting (RR) and secondary progressive (SP) multiple sclerosis

patients. p-values refer to the between-group comparison of the

Expanded Disability Status Scale change.

Esposito et al. 1493

even in the 1-year follow-up (69% in RR and 88% inSP MS patients, p< 0.001) (Table 3).

During MTX treatment, an annualized relapse ratereduction of 69% and 85% compared with the pre-MTX period was observed in RR and SP MS patientsrespectively (p< 0.001). This positive effect was main-tained also during the 1-year follow-up, without a sig-nificant difference between the two disease subgroups(p< 0.001) (Table 2).

MRI outcomes

A brain MRI scan was performed before and at the endof MTX treatment only in a subgroup of 64 RR and160 SP MS patients. The demographic and clinicalcharacteristics of this subgroup were not differentfrom the data observed in the whole population. Theproportion of patients with at least one enhanced lesionat baseline brain MRI was 66% and 53% in RR and SPMS patients respectively. After treatment, the propor-tion was 8% and 15% respectively, with a decrease of88% in RR MS and 72% in SP MS if compared withthe baseline MRI scan (p< 0.001) (Table 2).

Safety profile

The adverse events observed in our MS patients duringand after the treatment are reported in Table 4. Ninepatients (3.1%) developed a neoplastic disease. In par-ticular we observed two cases of acute myeloid leuke-mia: both patients were males and received nine cyclesof treatment. The diagnosis of therapy-related acuteleukemia (TRAL) was done after, respectively, 26 and33 months from the last MTX administration; thepatients received a cumulative dose of MTX of 65mg/m2 and 90mg/m2 respectively. The first patient devel-oped TRAL at the age of 33 years. He was treated withAIDA protocol19 and achieved a complete and persis-tent remission at a follow up of 22 months from thediagnosis of TRAL. The second patient developedacute leukemia at the age of 29 years. He was treatedwith the same scheme described above with completeremission for 23 months; then he developed carcinoma-tous meningitis due to the reactivation of the neoplasticdisease. He was treated with intrathecal chemotherapywithout benefit and he died about 2 years after theonset of TRAL. We also observed three cases of

Table 2. Clinical and neuroradiological characteristics of relapsing–remitting (RR) and secondary progressive (SP) multiple sclerosis

(MS) patients

RR MS patients (N¼ 79) SP MS patients (N¼ 210)

Baseline End of MTX

1 year

after MTX p-value Baseline End of MTX

1 year

after MTX p-value

Disability progression-freepatients (%)

34.2 94.9 5.8 <0.001 27.6 71.0 65.7 <0.001

EDSS (mean) 3.0 2.6 2.4 <0.001 5.3 5.7 5.9 <0.001

Annualized relapse rate(mean)

1.6 0.5 0.4 <0.001 0.6 0.1 0.1 <0.001

Patients with active brainMRI (%)*

66 8 NA <0.001 53 15 NA <0.001

The table illustrates the clinical and neuroradiological characteristics of RR and SP MS patients before the beginning of MTX, at the end of the

treatment and 1 year after the end of MTX.

*The MRI data are available for a subgroup of 64 RR and 160 SP MS patients. These data refer to the proportion of patients with at least one

Gd-enhanced lesion in a brain MRI performed within a 3-month window before and after MTX treatment.

NA, not applicable; MTX, mitoxantrone; EDSS, Expanded Disability Status Scale.

Table 3. Proportion of relapses in relapsing–remitting (RR) and secondary progressive (SP) multiple sclerosis (MS) patients

RR MS patients (N¼ 79) SP MS patients (N¼ 210)

2 years pre-MTX MTX 1 year follow-up p-value 2 years pre-MTX MTX 1 year follow-up p-value

0 relapses (%) 0 63 69 <0.001 41 82 88 <0.001

1–2 relapses (%) 43 30 30 <0.001 40 15 11 <0.001

>2 relapses (%) 57 7 1 <0.001 19 3 1 <0.001

The table illustrates the proportion of relapses in RR and SP MS patients. Specifically, patients were divided into three different categories, according to

the frequency of the attacks. The relapses were evaluated in three different intervals of time: the 2 years before mitoxantrone (MTX), the treatment

period and the 1-year follow-up. p-value refers to differences in the proportion of patients with no relapses, 1–2 relapses or >2 relapses during MTX

treatment and the 1-year follow-up period, compared with the 2-year period before the treatment.

1494 Multiple Sclerosis 16(12)

prostate cancer, two cases of breast cancer, one patientwith thyroid neoplasia and one patient with kidneycancer. We have a complete follow-up for four of theseven patients; they achieved a complete remission afterthe cancer treatment at a mean follow-up of 51 months.

Menstrual disorders were reported by 36.1% offemales: 15.1% had persistent amenorrhea, 16.1%had transient amenorrhea and 4.9% had menstrualirregularity. As regards the patients with persistentamenorrhea, the mean age was 40 years (median age:40.3 years, range: 28.6–50.0 years). Only 2.4% ofpatients showed sub-clinical cardiac toxicity, whichwas not clinically relevant. Finally, among the minorside effects, transitory nausea and vomiting, strictlyrelated to the first days after infusions, were reportedby 45.3% of patients and urinary infections werereported by 26.9% of patients.

Discussion

The efficacy of MTX was proven in the ‘90s, thanks tothree controlled clinical trials that led to the approvalof this treatment by the FDA and the EuropeanMedicines Agency (EMA). The French–British trial7

recruited RR and SP MS patients treated with MTX20mg and methylprednisolone 1 g monthly vs. methyl-prednisolone 1 g monthly alone for 6 months. Thisstudy showed an 86% decrease in the proportion ofpatients with enhanced lesions, a reduction of the pro-gression of disability by 84% and a reduction of relapserate by 77%. An Italian study,8 conducted in RR MSpatients, confirmed the efficacy of MTX 8mg/m2 vs.placebo every month over 12 months in the reductionof both disability progression (79%) and relapse rate

(60%). Finally, in a double blind, placebo-controlledtrial which evaluated the safety and efficacy of MTX5mg/m2 and MTX 12mg/m2 every 3 months in RR andSP MS patients (MIMS trial9), the administration of thedrug for 2 years led to a reduction of disability progres-sion in 64% of treated patients and to a reduction ofrelapse rate by 60%. Moreover, a post-hoc analysis ofthis study showed that this beneficial effect was sus-tained for at least 12 months after the end of the treat-ment.20 Unfortunately, both the French–British trialand the MIMS trial did not perform a comparativeanalysis to evaluate possible different treatmentresponses in RR and SP MS patients.

After MTX approval, several studies were conductedto confirm the efficacy and safety profile of the drug indifferent patient populations. A Sardinian study,21 per-formed on 45 highly active RR MS patients, showed asignificant improvement of the annualized relapse rateand of the Expanded Disability Status Scale score aftera mean follow-up of 3.6 years. This study reported alsoa statistically significant reduction of the MRI activityafter a mean follow-up of 1.8 years. A French retro-spective study conducted by Debouverie et al.,22 whichenrolled 141 subjects with active RR MS and 163 sub-jects with progressive MS, provided evidences of anoverall clinical improvement at the end of MTX treat-ment, more relevant in the RR subgroup. However,during the three-year follow-up period, both RR andSP MS patients underwent a progressive worsening ofExpanded Disability Status Scale score. Buttinelli andcolleagues23 described a significant effect of MTX onthe MRI activity in 25 RR MS and 24 SP MS patients;nevertheless, a confirmed disability progression in bothdisease subgroups was observed over a 5-year follow-up(8/25 in RR MS and 14/24 in SP MS patients). Finally,more recently, Le Page and colleagues evaluated thelong-term efficacy and safety of MTX in 100 consecu-tive patients with aggressive RR MS, who completed atleast 5 years of follow-up.24 In this cohort of aggressiveRR MS patients, relapse frequency and MRI activitywere reduced respectively by 91% and 89% (similar toour RR MS patients with, respectively, 69% and 88%reduction). These results were also associated with animprovement of Expanded Disability Status Scale scorein 64% of the patients at month 12 after the beginningof the treatment, with no additional progression ofExpanded Disability Status Scale score up to 5 yearsin 60% of the patients. It is important to observe thatthe studies conducted by Debouverie et al.22 andButtinelli et al.23 did not perform a comparative anal-ysis of MTX response between RR and SP subgroups,which is the aim of our study. In fact, while there are nomajor doubts about the beneficial effects of this immu-nosuppressive drug in RR MS patients, its use inpatients with SP MS remains more controversial.25

Table 4. Frequency of adverse events reported by patients

Adverse events Frequency

Nausea–vomit 45.3%

Menstrual disorders 36.1%

Urinary tract infections 26.9%

Hepatic enzyme alterations 4.1%

Alopecia 3.8%

Cardiac toxicity: 2.4%

– clinical cardiac abnormality 0.0%

– >10% reduction of LVEF on echocardiogram 2.4%

– LVEF <50% on echocardiogram 0.0%

Malignancies 3.1%

– acute leukemia 0.7%

– other cancers 2.4%

Asthenia 1.6%

Thrombophlebitis 1.0%

LVEF, left ventricular ejection fraction.

Esposito et al. 1495

We conducted this retrospective, non-randomized,open-label, observational study in order to evaluatethe clinical and neuroradiological response to thedrug in the two different disease subgroups.Moreover, we evaluated also the clinical parameters 1year after the end of the immunosuppressive treatment.All the analyses were performed comparing clinical andMRI parameters in RR and SP MS patients. The twosubgroups were comparable in terms of disability pro-gression during the 2 years before MTX treatment,whereas they significantly differed considering baselineExpanded Disability Status Scale score and the numberof relapses in the previous 2 years. A similar response inthe two subgroups of patients was observed for bothclinical and MRI disease activity parameters; in factboth RR and SP MS patients had similar positiveeffects on the reduction of annualized relapse rate andon the proportion of Gd-enhanced lesions after thebeginning of MTX. In contrast, when we consideredthe mean Expanded Disability Status Scale change, anopposite behavior in the two disease subgroups wasobserved (Figure 1). RR MS patients had a significantimprovement of the Expanded Disability Status Scalescore during the treatment period and this effect wasmaintained also at a 1-year follow-up evaluation. Anopposite trend was observed in the SP MS patients,who showed a continuous worsening both during andafter the MTX treatment. After dividing the SP MSpatients in two subgroups, based on the duration ofthe progressive phase at the beginning of MTX treat-ment, we observed that patients treated in the earlyphase of disease progression showed a trend for abetter response to the immunosuppressive drug in thefollow-up period, compared with patients treated later.These results could be explained by the mechanism ofaction of MTX, which is able to promptly reduce theinflammatory activity when it is present. Since the rel-evance of its efficacy seems to be related to the degree ofthe underlying CNS inflammatory activity, the clinicaland MRI beneficial effects were greater in RR MSpatients, who usually present more remarkable inflam-matory processes than SP MS patients.

Our results match previous experiences with cyclo-phosphamide26 and with a novel monoclonal antibody,alemtuzumab.27,28 Specifically, Coles et al.27 observed adissociation between the suppression of inflammationand the disease progression in 22 RR and 36 SP MSpatients treated with alemtuzumab. In fact, despite apositive effect on the clinical and MRI measures of dis-ease activity in both the two subgroups, disability wasdifferently affected depending on the phase of the dis-ease: SP MS patients showed sustained accumulation ofdisability, whereas in RR MS patients a reduction indisability was observed up to 36 months after alemtu-zumab. Moreover, in a phase II clinical trial,28

alemtuzumab demonstrated a greater effect, comparedwith interferon beta 1a, in reducing both the risk ofrelapse and the disease progression in patients affectedby RR MS, treated in the early phase of the disease.These data are consistent with what we observed in ourstudy and confirm the strong efficacy of intensiveimmunosuppressive therapies when the inflammatoryactivity is more prominent.

In order to define the real benefit–risk ratio, we alsocollected the adverse events developed in our cohortof patients during and after the treatment. The more fre-quently reported short-term adverse events were fatigue,alopecia, nausea, vomiting and an increased susceptibilityto infections, while in the long-term period MTX wasassociated to amenorrhea, infertility, cardiotoxicity andTRAL. A correct evaluation of the safety profile of thisdrug is hampered by the different treatment regimens usedin the different clinical trials or in the clinical practice.

So far, the most relevant and life-threatening side-effect related to the cumulative dosage of MTX is car-diotoxicity, which represents a definitive limitation inMTX use for a long period. An asymptomatic decreasein LVEF to <50% was reported in 5% of patientstreated with a cumulative dosage above 100mg/m2.13

However, early cardiotoxicity, even at lower dosage, aswell as late cardiac manifestations, also several yearsafter the end of MTX treatment, has been described.5,29

In our study, with our regimen of MTX use, only sevenpatients (2.4%) presented an asymptomatic LVEFdecrease >10%, with no patients having a LVEF<50%. We have to point out that most of our patientsdid not undergo an echocardiogram before each infu-sion, as recommended by FDA, because they weremainly treated before 2005 and at that time this examhad to be performed prior to the beginning of therapy,after six cycles and at the end of treatment.

In our study the safety data led to the raising of majorconcerns, since the number of patients who developedTRAL was higher than expected. A recent review of therisk of AL reported a 0.3% 3-year incidence of TRALfollowing MTX treatment.30 Compared with the risk inthe general population of developing acute myeloblas-tic leukemia, this data represents an approximately100 times increased risk.30 Moreover, it has beenobserved that the risk of developing TRAL can persistfor many years after the end ofMTX (up to 5 years aftertreatment withdrawal) and that follow-up studies aregenerally too short to correctly estimate the risk of thisadverse event.31,32 The frequency of TRAL in our studyis 0.69%; this means that, for every 145 patients treatedwith MTX, one develops AL. This value is twice that ofdata reported in literature. However, it is consistent withan ongoing, multicenter, retrospective study aimed atdefining the real incidence of TRAL in a large cohortof Italian patients treated with at least oneMTX infusion.

1496 Multiple Sclerosis 16(12)

Preliminary results showed a cumulative AL incidence of0.74% in MTX-treated patients,33 while the cumulativerisk of AL in the Italian population, with age lower than64 years, was about 1/1000. The two cases of TRAL weobserved were both cases of acute promyelocytic leuke-mia, which is the most common subtype of acute myeloidleukemia related to the treatment.30 Since the incidence ofTRAL in ItalianMS patients treated withMTX is higherthan reported previously, we suggest that all MTX-trea-ted patients should undergo a prolonged and carefulhematological follow-up.

We observed also a few cases of solid tumors in a pop-ulation of relatively young people; however, it is difficultto evaluate the real effect ofMTX in the onset of all thesedifferent subtypes of cancer. Moreover, comparing theprevalence of cancer observed in our populationwith the prevalence observed in an age-matched Italianpopulation, there is no evidence for an excess of prostateand breast cancer among our MS patients treatedwith MTX.34 These findings suggest that a thorough,post-marketing collection of safety data is warranted tobetter define the real carcinogenic risk ofMTX treatment.

Finally, menstrual disorders were reported by 36%of females; in particular 15.1% had persistent amenor-rhea and 16.1% had transient amenorrhea. These dataare consistent with literature data, which reported a14% of permanent amenorrhea in patients over theage of 35 years.35

In conclusion, MTX should be used in RR MSpatients who are non-responders to first-line immunomo-dulating treatments or as an induction therapy in patientswith evidences of active and aggressive disease since thebeginning. In fact, in these patients, MS is characterizedby a predominant inflammatory activity and, for thisreason, they could receive the strongest positive effectsby MTX administration. In contrast, we should considerthe use of this immunosuppressive drug only in SP MSpatients with evidences of disease activity and especiallyin the early phase of the progressive course, when theclinical worsening is still predominantly determined byan ongoing inflammatory activity. The efficacy of MTXindeed is definitely smaller when disability progression ismainly related to the degenerative processes. Therefore,in patients with SP MS, the benefit–risk ratio has to becarefully estimated on an individual patient analysis.

The results of our study, based also on the evidenceof an increased risk of serious adverse events, provideuseful information for everyday clinical practice. MSpatients have to be directly involved in the decisionprocess and they have to be correctly informed aboutthe occurrence and estimated likelihood of severeadverse events, as well as about the drug efficacy onthe disease. A recent work from Johnson et al.36 clearlydemonstrated that patients are willing to accept anincreased risk of serious adverse event, such as

TRAL, only if treatment is able to induce a strongeffect on disease activity and progression. Moreover,Cotte et al.37 recently described a possible approachto optimize the individual MTX benefit–risk profiledue to the discovery of pharmacogenetic markers.

Finally, the actual scenario is quite different from theone described in our study, due to the relatively recentapproval of natalizumab for the treatment of aggressiveRR MS. No comparative study is available at themoment between the two drugs and most of the patientstreated with natalizumab have a relatively short follow-up. The present paper strengthens the need for com-parative head-to-head observational studies, aimed toprospectively evaluate short- and long-term efficacyand safety of MTX and natalizumab. These studieswill contribute to better clarifying the subgroups ofMS patients with a more favorable benefit–risk ratio.

Funding

This research received no specific grant from any fund-ing agency in the public, commercial, or not-for-profitsectors.

Conflict of interest statement

– Dr. Federica Esposito, Dr. Marta Radaelli,Dr. Lucia Moiola, Dr. Filippo Martinelli Boneschi,Dr. Maria Assunta Rocca and Dr. MariaemmaRodegher report no disclosures.– Dr. Vittorio Martinelli has received speaker honor-aria and funding for travel from Biogen-Dompe SG,Merck Serono, Bayer Schering Pharma, Novartis andSanofi-Aventis; and has served as a consultant to BayerSchering Pharma, Sanofi-Aventis and TevaPharmaceutical Industries.– Dr. Maria Pia Sormani has received personal com-pensation for consulting services and for speakingactivities from Merck Serono, Teva, Actelion,Eidetica and Biogen Idec.– In the past year Prof. Giancarlo Comi has receivedpersonal compensation from Bayer Schering, SeronoSymposia International Foundation, Merck SeronoInternational, Sanofi-Aventis, Biogen Dompe and TEVAPharmaceutical Ind. Ltd and for speaking activities fromNovartis, Bayer Schering, Serono Symposia Int.Foundation, Merck Serono International, Sanofi-Aventis,Biogen Dompe and TEVA Pharmaceutical Ind. Ltd.

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