Multiple sclerosis (MS) is a common, immune-mediated, demyelinating disease that affects the central nervous system. MS has a highly variable course; therefore, patient-specific treatment decisions are becoming increasingly important. Currently, we have no way to differentiate between the patients who will acquire rapid disability progression and the ones who will remain stable over several years. Most studies point toward early intervention giving a better long-term outcome; however, long-term immunosuppression is associated with increased adverse risk [1,2]. Generally, MS therapies are long-term and some have a demonstrated rebound phenomenon precluding them from being stopped for long periods of time, such as fingolimod [3] and natalizumab [4]. However, there are currently 2 drugs that can potentially be used for immune reconstitution treatment: alemtuzumab and cladribine.

Alemtuzumab is administered in 2 courses, via infusion, 1 year apart. In the Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis Study (CARE-MS) extension trial, a redosing scheme was introduced, with up to 4 additional doses being offered in the event of recurring clinical activity [5]. Cladribine is a synthetic purine analog, and its mechanism of action is thought to be primarily via induction of apoptosis in lymphocytes [6]. It was shown in a phase III trial (A Safety and Efficacy Study of Oral Cladribine in Subjects with Relapsing-Remitting Multiple Sclerosis; CLARITY) to be highly effective in controlling disease activity [7-10]. With a no evidence of disease activity 3 (NEDA 3) rate of 44% over 2 years [7], it is placed in the range of the most efficacious disease-modifying therapies (DMTs) along with alemtuzumab (39%) [11,12] and ocrelizumab (47%) [13]. Furthermore, after 2 years of treatment and 2 years of follow-up (treatment free), 75% of patients with MS remained relapse free [8,14]. However, clinical stability beyond year 4, and additional doses of cladribine tablets based on clinical activity, has not yet been studied.

In the study discussed below, we will offer additional doses of cladribine tablets after 3 years. We aim to compare the clinical outcomes of patients with MS who received additional doses of cladribine with those who changed DMT. We also aim to identify biomarkers for disease control that can be used for treatment decisions, such as redosing with cladribine tablets versus change of DMT.

With the introduction of an ever-increasing number of DMTs, our treatment goals have shifted from simply reducing relapses to achieving NEDA.

In MS, NEDA 3 is defined as no clinically confirmed progression, no relapse, and no new or enlarging or gadolinium (Gd)-enhancing lesions [15]. This has since been expanded to NEDA 4, which includes brain atrophy [16]. Although our current treatments are increasingly effective, only 30% of patients with MS reach NEDA 4 after 2 years [17]. The rate of NEDA 4 in patients with MS treated with cladribine has not yet been established.

Magnetic resonance imaging (MRI) has been established as the most reliable indicator of long-term outcomes. Sormani et al [18] analyzed data from 13 large clinical trials, including 13,500 patients with MS, to show that new T2 lesions can predict disability progression. This prediction was significantly improved when combined with brain atrophy [18]. Some of the high efficacy treatments can change the rate of brain volume loss to that seen in the normal aging population [19]. As MRI technology advances, it has become clear that some substructures such as thalamic volume, lateral ventricle, and gray or white matter volume might be even more sensitive and correlate better with clinical outcomes [20].

Blood-based biomarkers are attractive because of their ease of collection and cost-effectiveness. In addition, they may better reflect or even predict disease activity. Lymphocyte subsets may be good markers of disease stability, as many currently used therapies act by restoring the balance of immune cells in the periphery to a healthy state. Therapies affecting the B-cell population have been described as effective treatments for MS [21]. This may be related to the suppression of memory B cells. This theory is supported by the failure of Atacicept and tumor necrosis factor α inhibitors in treating MS, both of which result in disease activation [21]. In both ORACLE-MS and CLARITY, cladribine has been shown to markedly reduce B cells, while having a more modest reduction in T cells and natural killer cells [22,23]. A more recent study of the CLARITY cohort further characterized this reduction and demonstrated that memory B cells were the predominantly affected subtype [24].

These studies investigated lymphocyte subsets over the course of 1-2 years. Therefore, a long-term investigation of lymphocyte subsets in response to cladribine treatment is warranted.

Cognitive impairment is a prevalent symptom in MS, with rates of approximately 40%-70% [25]. The severity of cognitive deficits varies, but unlike the physical symptoms associated with MS, cognitive deficits are unlikely to remit and are associated with a higher risk of progression [26-28]. A recent systematic review evaluated the literature on cognitive impairment and employment status and found a consensus that patients with MS who are unemployed or have reduced work hours record weaker cognitive scores [29]. Loss of employment is a major concern for patients with MS, particularly as the disease is often diagnosed in people of working age, who are just establishing careers and families. Despite this, cognitive testing is often disregarded in terms of clinical trial outcomes. Several tools have been developed to evaluate cognitive function, including the Brief Cognitive Assessment for Multiple Sclerosis (BICAMS) [30]. The BICAMS is quick and easy to administer and has recently been validated in the Australian population [31].

Epigenetics is a rapidly developing area of medical research and refers to the potentially reversible regulation of genomic functions, particularly gene expression. This provides a mechanism whereby an organism can dynamically respond to a change in its environment (eg, DMTs) and alter its gene expression accordingly. DNA methylation is a well-characterized, relatively easy to study epigenetic modification and generally refers to the addition of a methyl group to a cytosine base, followed by a guanidine (referred to as a CpG dinucleotide).

We, and others, have described differential DNA methylation profiles between healthy controls and patients with MS, as well as between relapsing remitting MS and secondary progressive MS [32-38]. In addition, we and others have performed longitudinal studies that found epigenetic profiles are altered after dimethyl fumarate treatment [39-41], making it a potentially specific biomarker for treatment response.

Neurofilament (NfL) light chains are found in neuronal cells but are shed into the cerebrospinal fluid upon neuronal damage and are detectable in the peripheral blood. Increased serum NfL levels have been identified in patients with MS compared with that in healthy controls [42]. These levels show a strong correlation not only with cerebrospinal fluid NfL levels but also with the presence and activity of focal lesions and clinical outcomes [42]. This makes them a promising biomarker not only of disease activity but also disease progression. These may be useful indicators of the need for additional courses of treatment.

The primary objective of the study is to evaluate the safety and efficacy of cladribine tablets over a 6-year period according to NEDA 3. We will also evaluate clinical outcomes and cognition over a 6-year period, blood-based and MRI-based biomarkers for their ability to predict treatment response, disease activity, and the need to redose cladribine subsequent to the 2-year, 2 initial courses.

This study is a multicenter, 6-year, phase IV, low interventional trial. Enrollment of 150 patients with MS was planned across 9 specialist MS clinics in Australia. The Therapeutic Goods Australia approved a cumulative dose of cladribine (10 mg) tablets as 3.5 mg/kg body weight over 2 years administered as 1 treatment course of 1.75 mg/kg per year. After 3 years, patients with MS in consultation with their health care provider will discuss the option of redosing with cladribine tablets, if clinically indicated (by relapse or new MRI activity), switch to another DMT, or continue without change (without commencement of any DMT). For those who will be continued on cladribine, additional courses will be administered as per the previous dosing of 1.75 mg/kg per year.

Patients must meet the criteria as described below (Textbox 1).

The primary end point for all outcome measures is the proportion of patients with MS achieving NEDA status (responders) at 6-years relative to screening. However, preliminary end points will also be considered at each of the major biomarker collection points (months 7, 24, 48, and Tv).

The key outcome measures will be NEDA 3 or 4 responders after 6-years. Additional outcome measures will change over time from baseline in MRI parameters (brain volume loss, lesion load, and lesion volume), cognition, lymphocyte subsets, sNfL, and global DNA methylation profiles. In addition, the Tv time point will be used to determine if there are any changes in biomarker status that may predict disease activity. This will be compared with the time point at which the patient is deemed to have stable disease.

This study was funded in October 2018 as an investigator-initiated trial by Merck Healthcare Pty Ltd, KGgA, Darmstadt, Germany, to the lead site (John Hunter Hospital).

This study is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12619000257167) with Universal Trial Number (U1111-1228-2165). Ethical approval for conducting this study was granted on November 8, 2018, by the Hunter New England Local Health District Human Research Ethics Committee (protocol 2019/ETH08849). The study will be conducted in accordance with the revised Declaration of Helsinki, and all participants will provide written informed consent to participate in this study.

As of May 2021, 145 participants have been enrolled in the study. Recruitment was completed by June 2021, and the study is expected to conclude in 2027.

Our study is designed to collect real-world outcomes, while filling the gaps of the previous randomized controlled trials (RCTs) of cladribine tablets for the treatment of MS. This study also offers the opportunity to assess the long-term use of cladribine tablets in a clinical setting and the effectiveness of additional courses of cladribine tablets based on disease activity, in ongoing treatment of MS.

RCTs are the gold standard for evaluating treatment outcomes and providing efficacy data for new treatments. However, the strict inclusion criteria and protocol-driven approach may lead to low generalizability because these trials are not always reflective of real-life care [47,48]. In addition, the frequency of MRI in MS RCTs is often higher than that in routine clinical practice [47,48]. Real-world studies can complement data from RCTs by investigating patients who are receiving treatment and being monitored according to routine clinical practice [47,48], and the importance of real-world clinical data is becoming increasingly recognized. For example, MSBase, which is now following over 70,000 patients with MS worldwide with regular assessments, has shaped clinical decision-making in the MS field over the past 5 years using real-world data [49].

The exclusion criteria for this study are deliberately minimal, so that most patients with MS who are planning to commence or choose to take cladribine tablets as part of their routine MS clinical care, are eligible to participate. This study imposes no limits on prior DMT exposure or immunosuppression, no upper limit on EDSS, and an upper age limit of 70. These relaxed inclusion criteria will allow us to capture the broadest range of patients with MS possible, and with the generation of data sets that are reflective of real-world MS demographics.

In the CLARITY study, 75% of the participants were treatment-naïve before entering [7]. As with all pharmaceutical trials, there were restrictions on prior DMT use (no more than two failed DMTs were allowed before study entry, and anyone who had used prior immunosuppression was excluded) [7]. This leaves a scarcity of data relating to how patients with MS who have been on prior DMTs, particularly immune suppressants with long-term effects such as alemtuzumab or even dimethyl fumarate, will respond to cladribine tablets. In addition, this does not reflect clinical reality, where many patients with MS have trialled several treatments and often failed several other DMTs before starting cladribine tablets. Data on the safety and effectiveness of cladribine after other DMTs are needed for clinicians and patients with MS to be able to make appropriate decisions about therapy, particularly if they are switching from a prior immunosuppressant. Our study is well-positioned to provide such data.

This trial will also investigate the effectiveness of additional courses of cladribine tablets based on disease activity. Alemtuzumab, another immunomodulatory therapy, is administered in a similar dosing scheme to cladribine tablets [12]. In the CARE-MS II trial, up to four additional courses of alemtuzumab were given to patients with MS as required, with success [5]. Starting in year 3, if patients with MS in this trial have a clinical or radiological relapse, they will be offered an additional course of cladribine. Although the CLARITY extension trial found that additional courses provided no additional benefit, these courses were not offered based on disease activity [9]. This will be the first investigation into an additional cladribine dose based on disease activity.

There is evidence that clinicians are shifting their treatment goals away from relapse free to achieving NEDA status. For example, a recent study using MSBase data demonstrated that just one new subclinical T2 lesion was associated with 1.62 times odds of changing treatment compared with no new lesions [50]. Patients with MS taking cladribine tablets achieved 47% NEDA 3 rates in CLARITY after 2 years [7]; however, rates of NEDA 4 and data beyond 2 years have not been reported. The addition of accelerated brain volume loss to the NEDA criteria (NEDA 4) is an important parameter as it has been shown to be predictive of long-term disability progression and cognitive decline [26,51]. This is highlighted in the FREEDOM trials, where 31% of patients with MS on fingolimod sustained NEDA 3 status, but only 19.7% achieved NEDA 4 after 2 years [16].

Achieving NEDA status may also differ outside the bounds of RCTs. A real-world study conducted in the USA (MS-MRIUS) evaluated nearly 600 patients with MS on fingolimod and found that NEDA 3 was achieved in approximately 58.7% of patients with MS, and 37.2% had achieved NEDA 4 [52]. The differences may be because of the shorter follow-up time (16 months vs 24 months), different patient populations (less severe disease course), or may be reflective of the real-world nature of the data collection versus an RCT [52]. Another real-world study also reported slightly higher levels of NEDA 3 (44%) after 2 years, but did not evaluate NEDA 4 [53]. There has been one small study of cladribine tablets from MSBase data, which compared outcomes over 1 year [54]. Although this study was shorter than CLARITY, the data were similar, with effects lasting at least 4 years [54] despite the majority of patients with MS receiving only one course of treatment. The MSBase study did not specifically report on NEDA 3 outcomes; therefore, it will be interesting to see if NEDA rates remain the same for cladribine use in the clinical setting.

There have been recent criticisms of the current NEDA criteria [55-57]. Stangel et al [57] suggested an algorithm that incorporates cognitive (SDMT), patient-reported outcomes, depression and anxiety ratings, and other parameters. Other criticisms suggest that biomarkers of inflammation and neurodegeneration in body fluids need to be added for a true reflection of disease activity (NEDA-5 or minimal evidence of disease activity) [56]. The inclusion of blood-based biomarkers in this study, including sNfL, as well as cognitive assessments (the BICAMS includes the SDMT) will ensure that we are also able to assess disease activity based on these parameters and assess NEDA5 or minimal evidence of disease activity when a consensus is reached. Furthermore, the discovery of biomarkers that may indicate a response to treatment may allow us to prevent patients with MS from undergoing life-long immune suppression if not required. This could be translated to other MS therapies.

There are minimal real-world data on the effectiveness of cladribine tablets for MS. This study will not only fill this knowledge gap, but also evaluate the efficacy of a clinically indicated additional course beyond the year 2 course. The regular collection of biospecimens for biomarker assessment will help identify biomarkers that may be indicators of treatment response and the need for additional dosing. This may help move clinical practices toward individualized dosing schedules.

Deidentified individual data sets will be available upon request to the authors following the publication of the results of the study.