Therapeutic choices and disease activity after 2 years of treatment with cladribine: An Italian multicenter study (CladStop)

Cladribine tablets, a purine analogue antimetabolite, offer a unique treatment regimen, involving short courses at the start of the first and second year, with no further treatment needed in years 3 and 4. However, comprehensive evidence regarding patient outcomes beyond the initial 24 months of cladribine treatment is limited.


INTRODUC TI ON
In recent years, there has been a radical change in the therapeutic scenario of multiple sclerosis (MS).The new knowledge of the disease has enabled the development of new disease-modifying therapies (DMTs), with different mechanisms of action, greater efficacy, and different safety and tolerability conditions.The current generation of DMTs include a wide range of immune modulators, immune depletion, and repopulating agents [1].
Such treatment comprises cladribine tablets, a small purine analogue antimetabolite that mimics adenosine, inhibits the action of the adenosine deaminase enzyme, and causes an interim reduction of lymphocytes with predominance in B cell and T cell counts followed by reconstitution of adaptive functions [2].
In 2017, cladribine was licensed by the European Medicines Agency as the first oral pulsed therapy for the treatment of adult patients with highly active relapsing MS [3].
Each tablet contains 10 mg cladribine, and the recommended total dose is 3.5 mg/kg body weight administered through two short courses at the beginning of the first and second year.Following completion of the two courses, no further cladribine treatment is required in years 3 and 4, but it is not contraindicated (summaries of product characteristics (SmPCs) Mavenclad April 2022).During the clinical trials, repeating the dose routinely beyond year 2 was not associated with significantly improved disease control (SmPCs Mavenclad April 2022) [4].
This treatment-free window offers different opportunities compared to continuous therapy regimens, such as patients' flexibility in family planning and attenuating or active vaccine administration [5], albeit real-world data confirm a normal humoral and cell-mediated immune response to vaccination following cladribine treatment regardless of the time of application and the lymphocyte count [6,7].
Different phase IV studies are ongoing aimed at collecting new evidence for further characterizing cladribine tablets' benefit/risk ratio.
Several postapproval data are arising from international realworld cohorts and have well characterized the efficacy and safety of the first 2 years of treatment with cladribine tablets [8][9][10].
Furthermore a post hoc analysis of the CLARITY study already supported few years ago the continuation of treatment in year 2 even if a patient reported clinical disease activity in year 1, as most of them experienced benefit from treatment dosing completion [11].
However, the evidence on the follow-up at 2 years of treatment with cladribine tablets meant as the end of 24 months and beyond is limited.This analysis aims to investigate the therapeutic choices and clinical disease activity of patients who completed a 2-year course with cladribine.

Study design and data collection
This is a multicenter, retrospective observational study that enrolled patients with MS who had completed 2 years of treatment with cladribine, with an observational follow-up period of at least 6 months.
The baseline for this study is defined as the completion of 2 years of treatment with cladribine, which corresponds to 24 months from the therapy start and is referred to as "CladStop" in this study.

Demographic characteristics, information about comorbidities
and prior infections, and data related to the patients' MS history were collected.Additionally, clinical, laboratory, and magnetic resonance imaging (MRI) variables (in terms of gadolinium enhancing lesions and new T2 lesions) obtained during the 2 years of treatment with cladribine were retrieved from medical charts.These variables include lymphocyte count, Expanded Disability Status Scale (EDSS) scores, MRI lesion counts, and relapse information.Data on the therapies initiated after the 2-year course of cladribine treatment were also collected.
MRI data, relapse occurrences, and changes in EDSS scores were collected at 6 and 12 months after CladStop.The evaluation of disability progression was considered in terms of an increase of at least 1 point on the EDSS if the baseline EDSS was equal to or less than 5.5, or 0.5 points if the EDSS was greater than 5.5.
Any relevant adverse events that occurred from the start of treatment with cladribine were documented in the electronic case report form.
Study data were collected and managed using REDCap electronic data capture tools hosted at University of Genoa [12].(iii) automated export procedures for seamless data downloads to common statistical packages, and (iv) procedures for data integration and interoperability with external sources [13].

Outcomes
The primary outcomes of this study are the therapeutic choices and clinical disease activity assessed by the annualized relapse rate (ARR) following the completion of the 2-year treatment course with cladribine.
The choice of ARR as the primary outcome is supported by its widespread use as an important measure in clinical trials evaluating the efficacy of treatments for MS [14,15], such as cladribine.These trials have shown promising results, with a significant decline in relapse rates observed after the initiation of therapy [16,17].

Statistical analysis
Continuous variables were described using mean and SD or median and range or interquartile range; categorical variables were reported as counts and percentages.
The time to initiate a new treatment after CladStop was assessed using a Kaplan-Meier curve and analyzed using a multivariable Cox regression model, which was adjusted for baseline characteristics.
Differences in ARR over time were determined with Friedman test and a post hoc pairwise comparison using the Bonferroni correction.
Significance level was set at 5% for all analyses, and all tests were two-tailed.

Ethics statement
The study was conducted in compliance with the principles of the Declaration of Helsinki.The protocol was approved by the regional ethics committee (CER Liguria: 273/2022-DB id 12,395, 20 June 2022).Written informed consent was obtained from all participants before starting any study procedures.

RE SULTS
A total of 204 people with MS (pwMS) from 17 Italian and one Swiss center were enrolled between June 2022 and May 2023.

Demographic and clinical characteristics are shown in Table 1.
The mean age at CladStop was 36.6 (SD = 10.45) years with a range of 18 to 68 years and a predominance of female patients (70.6%).Approximately three quarters of the patients (76.0%) had no comorbidities (Table 2).The median observation period after CladStop was 17.5 months, ranging from 6 (minimum allowed) to 36 months.At 12 months, the probability of starting a new treatment was 12.1%, which increased to 24.6% at 24 months.Among all participants, 37 individuals (18.1%) started a new treatment following cladribine therapy.Table 3 presents the characteristics of these patients.The most common treatments were ocrelizumab (n = 15, 7.4%) and natalizumab (n = 8, 3.9%).No patients received an additional cycle of cladribine during the entire follow-up as per Italian rules (Figure 1).Nineteen patients showed MRI activity (data not shown).
The ARR improved significantly as early as the first year of treatment (0.12 ± 0.37, p < 0.001), confirmed after the second year (0.13 ± 0.52, p < 0.001); the ARR further decreased after 12 months of follow-up after CladStop (0.07 ± 0.25, p < 0.001), again compared with the ARR in the year prior to cladribine start (0.82 ± 0.80; Figure 2).
In terms of lymphocyte counts, the trends over the 2 years mirror each other, with a marked increase in lymphopenia (grade III or higher) immediately after the treatment cycle (3.6% in the first year and 8.8% in the second year), followed by a recovery at month 7 (1.8% in the first year and 2.6% in the second year).This trend is particularly noticeable in the second year (Figure 3).The presence of high grade of lymphopenia was consistent among all patients throughout the entire study period, regardless of their age, except during the third month of treatment in the second cycle, when a higher frequency of events was observed in the elderly group (>50 vs. ≤50 years old: 26.3% vs. 6.2%, p = 0.004).
Eighteen (8.8%) patients presented at least one adverse event (AE) for a total of 23 cases.Twenty AEs occurred in 15 patients during the 2 years of treatment and three in three patients in the 12 months following CladStop.One serious AE, prostate cancer, occurred in one pwMS during the 12-month following CladStop; this AE was considered not related to the treatment by the treating neurologist (Tables 5 and 6).Three cases of oral herpes, indicated by physician as probably or certainly related to cladribine use, were reported during 2 years of treatment.
They were mild and resolved within a few days; these patients did not have lymphopenia.However, it is worth mentioning that prophylaxis for herpes zoster is recommended for patients with grade 4, for grade 3 lymphopenia, or for immunocompromised individuals [18].

DISCUSS ION
This study reports on the therapeutic choice at the end of 2 years of treatment, the evidence of cladribine's effectiveness in reducing relapse rates and disability progression, and the drug's safety profile.

TA B L E 3 (Continued)
Overall, cladribine showed a favorable safety profile consistent with its mechanism of action [19]; lymphopenia (grade ≥ 3) was reported in a subgroup of patients immediately after each cycle of cladribine, both during the first year (about 4%) and during the second year (about 9%).Lymphocyte counts returned to a normal range as we moved away from the last dose (at the seventh month of treatment), consistent with data from the literature [2].
Noticeably, two of three cases of oral herpes occurred in nonelderly adult patients coming from a previous DMF treatment, whereas one was not previously treated.This aligns with recent literature [20], which reported that herpes infections predominantly occurred in patients who had received DMF as prior therapy.
After CladStop, only three AEs were recorded in three patients.
Among these, only one case of moderate severity gingivitis was reported as possibly related to cladribine and occurred 7 months after 2 years of treatment.
These results are consistent with other reports based on real-life data, confirming that ocrelizumab and natalizumab are the primary choices for switching after 2 years of treatment with cladribine [21].
Additionally, they indicate a reduction in ARR during and after treatment compared to the pretreatment period, as well as a predominance of mainly mild to moderate adverse events [16].Finally, they demonstrate a similar rate of improvement in EDSS after the end of 2 years of treatment [22].This study has limitations.To begin with, it is important to note that the study is observational and retrospective.There is a possibility that physicians might not have accurately reported specific details, and the lack of clinical evaluations at certain time points could affect the overall reliability of the findings.
Additionally, the chosen follow-up period after the completion Data Capture) is a secure, web-based software platform designed to support data capture for research studies, providing (i) an intuitive interface for validated data capture, (ii) audit trails for tracking data manipulation and export procedures, | 3 of 13 CladStop: ITALIAN MULTICENTER OBSERVATIONAL STUDY WITH CLADRIBINE Before starting cladribine treatment, patients had a median EDSS score of 2.0 (with an interquartile range of 1.0-3.0),and on average, they had undergone one (range = 0-6) previous DMT (the most common was dimethyl fumarate [DMF], 29.2%).Most patients (n = 128, 62.7%) experienced at least one relapse in the 12 months before starting cladribine, and 136 individuals presented MRI activity (data available on 164 pwMS).
overall significant improvement in the ARR at the 12-month follow-up after CladStop compared to the pretreatment period and the first 2 years of treatment.This observation suggests a concrete reduction in the occurrence of relapses in pwMS irrespective of their treatment choice after the completion of the second cycle of cladribine.The analysis of time to starting a new treatment after CladStop reveals two significant associations (both p < 0.001) and provide insights into the factors influencing treatment decision following 2 years of treatment with cladribine.First, patients who experienced relapses during the 2 years of cladribine treatment demonstrated a threefold higher probability of initiating a new therapy after CladStop.Second, there is an inverse proportional relationship between patients' age and the likelihood of starting a new therapy after CladStop.However, most of the patients did not initiate new treatments during the 12 months following the completion of the treatment with cladribine, suggesting a stable clinical status that did not require subsequent additional therapies.Only a small proportion of patients (9%) showed MRI activity and/or worsening on the EDSS (18.8% in 143 patients with evaluation at 12-month follow-up).

F I G U R E 1
Therapeutic choice after CladStop.DMT, disease-modifying therapy.TA B L E 4 Factors associated with starting a new therapy after CladStop.

of 2 F I G U R E 2 F I G U R E 3 | 9 of 13
years of cladribine treatment is relatively short (a minimum of 6 months and a maximum of 36 months).A longer term follow-up would provide a more comprehensive understanding of the sustained effectiveness and safety profile of cladribine beyond the immediate posttreatment period.Finally, the study primarily focuses on clinical outcomes, such as relapse rates and disability progression, but does not extensively explore or collect information about patient preferences or factors influencing their subsequent therapeutic choices.A more comprehensive understanding of patient perspectives, for example, through patient-reported outcomes, could enhance the interpretation of treatment decisions.Annualized relapse rate before, during, and after cladribine.* indicates statistically significant.Lymphocyte count during 2 years of treatment.CladStop: ITALIAN MULTICENTER OBSERVATIONAL STUDY WITH CLADRIBINE TA B L E 5 Safety data.

Table 4
shows the analysis of time to starting a new treatment after CladStop.Patients who experienced relapses during the 2 years of cladribine treatment had a higher probability of starting a new therapy after CladStop (hazard ratio [HR] = 3.78, 95% confidence interval [CI] = 1.82-7.85,p < 0.001) compared to patients without relapses.Furthermore, the probability of starting a new therapy decreased with the age of the patients (HR = 0.68, 95% CI = 0.47-0.98,p = 0.039).Patients who had higher disease activity before cladribine, as indicated by MRI activity or previous relapses, did not exhibit a significantly higher probability of initiating new therapy compared to other patients.
Note: Data are presented as n (%).Abbreviations: AE, adverse event; SAE, serious AE.TA B L E 6