Nilotinib treatment outcomes in autosomal dominant spinocerebellar ataxia over one year

Study population

From an ongoing institutional cohort of patients with cerebellar ataxia, 108 subjects with cerebellar ataxia who visited the Neurology Department of Seoul National University Hospital between May 2019 and March 2020 were initially included. Among them, the final study population included patients meeting the following criteria: (1) patients without a chronic heart, liver, kidney, or hematologic disorder that restricts the use of nilotinib; (2) patients who started regular administration of nilotinib with available baseline evaluations for ataxia severity; (3) patients whose ADSCA was confirmed as one of SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, and SCA17^1,2; and (4) patients who completed 1-year regular follow-up evaluations for ataxia severity^17,18,21. The decision to start nilotinib treatment was made by the subjects and their caregivers after they were fully informed about the adverse effects and cost of nilotinib by the treating physician (K.C.). Before treatment initiation, checks for chronic cardiac, hepatic, or renal disorders were performed by evaluations of the subject’s medical history and laboratory parameters, including the complete blood count, liver enzyme panel, blood urea nitrogen and creatinine, serum electrolyte panel, and antibodies for chronic hepatitis viruses, and electrocardiography^8,17,18,20. The dosage of nilotinib ranged from 150 to 300 mg per day, and the dosage was adjusted by the treating physician (K.C.) if clinically indicated, according to the clinical responsiveness and the subject’s tolerance to the medication^17,18,20.

Genetic testing for ADSCA

Genetic testing for ADSCA was performed using subjects’ serum with PCR analysis for the abnormal expansion in CAG repeats in the genes ATXN1, ATXN2, ATXN3, CACNA1A, and ATXN7; CAG/CTG repeats in ATXN8; and CAG/CAA repeats in TBP. For each gene, the cutoff for the trinucleotide repeat number for the full penetrance of disease was set as 39 for ATXN1, 37 for ATXN2, 60 for ATXN3, 20 for CACNA1A, 37 for ATXN7, 54 for ATXN8, and 49 for TBP^1,2.

Acquisition of clinical and laboratory data

Along with demographic information, clinical and laboratory information, including the age at the onset of the clinical symptoms of cerebellar ataxia, maintenance dosage of nilotinib, and the length of trinucleotide repeats in the allele of longer repeat length, were obtained. The severity of ataxia and the ability to perform activities of daily living (ADLs) were assessed at baseline and at 1, 3, 6, and 12 months (− 1 M, − 3 M, − 6 M, and − 12 M, respectively) after the initiation of nilotinib administration. At every follow-up visit, the subjects were monitored for adverse drug reactions to nilotinib and underwent evaluations for a complete blood count, liver enzyme panel, blood urea nitrogen and creatinine, and serum electrolyte panel as well as electrocardiography^8,17,18,20.

The clinical severity of ataxia was assessed using the Scale for the Rating and Assessment of Ataxia (SARA) scores (8 items; score range, 0–100, where a higher score indicates a higher severity of ataxia)^17,18,22. The SARA score was measured by two neurology experts (W.-J.L. and Y.-H.J. or Y.-W.S.). For the discrepant cases, consensuses were reached after a review of the videotape and discussion. Inter-rater (W.-J.L. and Y.-H.J. or Y.-W.S) agreement for SARA score assessed through separate reviews of the video recordings for the SARA score evaluation at each time points was high (0.82). The changes in ADL functioning were assessed using a battery of structured questionnaires, which included the functional stage subscale of the Friedreich Ataxia Rating Scale (FARS I; score range, 0–6, with a higher score indicating a more advanced stage of disease), the ADL subscale of the FARS (FARS II; score range, 0–36, with a higher score indicating more impaired ADL functioning), and the modified version of the Barthel Index (BI; score range, 0–100; with a higher score indicating a higher degree of independence)^23,24.

Outcome analysis

The main outcome was the change in the SARA score from baseline. Subjects were categorized into the ‘responsive’ group when the SARA score change-12 M (SARA score at baseline minus the SARA score at 12 M) was > 0 or into the ‘nonresponsive’ group when the SARA score change-12 M was ≤ 0. The changes in the FARS I, FARS II, and BI scores were also measured. Adverse events were classified according to the Common Terminology Criteria for Adverse Events (CTCAE v5.0)²⁵. A severe adverse event was designated as an adverse event of CTCAE grade 3 or higher.

Serum proteomic analysis

Five subjects with the highest SARA score change-12 M scores in the responsive (R) group and five subjects with the lowest SARA score change-12 M scores in the nonresponsive (N) group were selected for the serum proteomic analysis. Serum samples were obtained at the time of laboratory check-up before the initiation of nilotinib treatment. As a control (C) group, five subjects with recurrent dizziness and a similar age range to those of the patient groups but without evidence of ataxia or chronic central nervous system disease were selected from our serum repository database^26,27.

Serum proteomic analysis was performed using 200 µl of serum samples stored at − 80 °C. The detailed process of proteomic analysis was described in previous reports^28–30. In brief, the process included serum sample preparation, liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis using a Q-exactive HF-X (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Ultimate 3000 RSLC system (Dionex, Sunnyvale, CA, USA), and data processing for label‑free quantification. Mass spectra were processed in MaxQuant software (ver. 1.6, Max-Planck Institute, Munich, Germany), and MS/MS spectra were searched against the Human UniProt protein sequence database. The required false discovery rate (FDR) for all proteins and peptides was set as 1%.

The analysis of the differentially expressed proteins (DEPs) was performed using Perseus software³⁰. The expression level of proteins in the serum was estimated by the intensity-based absolute quantification values using MaxQuant software. After log2 transformation of those values, proteins with less than three quantified values in each group were filtered out, and missing values were imputed based on a normal distribution (width = 0.3 and downshift = 1.8). Analysis of variance (ANOVA) was performed to detect DEPs among the groups with a significance level of P < 0.05. DEPs were z-normalized for their abundances followed by hierarchical clustering with Euclidean distance. For the adjustment for multiple comparisons, the Benjamini‒Hochberg method was used with the Benjamini‒Hochberg critical value calculated using the false discovery rate of 0.2³¹.

Statistical analysis

R software version 4.0.3 (2021; R team, Vienna, Austria) was used for all statistical analyses. Data are reported as numbers (percentage), mean ± standard deviation, or median (range). For the comparison between responsive and nonresponsive groups, a two-tailed Student’s t test, Pearson’s chi-square, Kruskal‒Wallis, or Mann‒Whitney test were used. Spearman’s rho was used for the correlation analyses between the SARA score and the FARS I, FARS II, and BI scores. To evaluate the change in the SARA, FARS I, FARS II, and BI scores from baseline, repeated-measure analysis of covariance (RM-ANCOVA) adjusting for age, sex, type of SCA, baseline score, duration of disease, maintenance dosage of nilotinib, and length of the trinucleotide repeat was performed. For every analysis, a P value of < 0.05 was considered statistically significant.

Standard protocol approvals, registrations, and patient consents

The study procedures were approved by the institutional review board of the Seoul National University Hospital. All methods were performed in accordance with the STROBE guidelines for cohort studies, and the 2013 amended version of the Declaration of Helsinki. The protocols of the study were registered in clinicaltrials.gov (Effect of Nilotinib in Cerebellar Ataxia Patients, registration number: NCT03932669, date of registration: 01/05/2019). Written informed consent was obtained from all study participants. The study protocol and statistical analysis plan are available in Supplemental Dataset.

Patient characteristics

Among the initially included 108 subjects with cerebellar ataxia, 70 subjects without a chronic heart, liver, kidney, or hematologic disorder and with available baseline evaluations for ataxia severity and started regular administration of nilotinib. Among them, 18 subjects without a confirmed diagnosis of ADSCA, three subjects with a trinucleotide repeat number of reduced-penetrance trinucleotide alleles, and 17 patients who did not complete 1-year maintenance therapy with nilotinib and regular follow-up evaluations were sequentially excluded, and the remaining 32 subjects (14 [43.8%] males and 18 [56.2%] females, median age 42 [30.5–49.5] years) were included in the final analysis (Fig. 1). Eight (25.0%) subjects had SCA2, 10 (31.2%) had SCA3, 8 (25.0%) had SCA6, 3 (9.4%) had SCA7, 2 (6.2%) had SCA 8, and 1 (3.1%) had SCA 17. The median age of onset was 44 [31–50] years, and the median duration of disease from symptom onset was 4.5 [2–8] years. At baseline, the median SARA score was 10.5 [8.8–13.2], the median FARS I score was 2 [2], the median FARS II score was 9.5 [6.5–13], and the median BI was 1.5 [0–3].

Efficacy of nilotinib in ataxia symptoms

The maintenance dosage of nilotinib was 150 mg/day in six (18.8%), 200 mg/day in three (9.4%), and 300 mg/day in 23 (71.9%) subjects. The median SARA score change-12 M was 1.5 [− 0.5–2.2], and 20 (62.5%) subjects were categorized as responsive. The demographic characteristics, frequencies of SCA types, trinucleotide repeat length, onset age, baseline scores for ataxia severity, and maintenance dosage of nilotinib were similar between the responsive and nonresponsive groups (Table 1). Among the 17 subjects who did not complete follow-up, the reasons for discontinuation were a lack of subjective improvement in ataxia symptoms in four subjects, adverse events in three subjects (two with dizziness and one with stomach pain, all CTCAE grade 1), and the cost of the drug in three subjects, while in the remaining seven subjects, the reason was not identified.

Although the SARA score at baseline exhibited a moderate correlation with the baseline scores of the FARS I, FARS II, and BI (all, P < 0.001), the correlation of the change in the SARA score with the changes in those scores was weak (Supplemental Table 1 and Supplemental Fig. 1). Adverse events were observed in 15 (42.9%) subjects, nine with transient and mild alanine aminotransferase increases, two with gastrointestinal discomfort, one with mild folliculitis, one with urticaria, one with dizziness, and one with headache (all CTCAE grade 1).

SARA score at 3 M exhibited a significant improvement compared to the baseline and to 1 M (P = 0.011 and P = 0.001, respectively) and SARA score at 6 M showed a higher improvement compared to the 1 M (P = 0.041). However, SARA score at 12 M did not exhibit a significant improvement compared to the baseline (P = 0.058, Fig. 2A). In RM-ANCOVA, the SARA score showed significant improvement from baseline (F = 2.595, P = 0.039), and higher SARA scores at baseline (F = 589.207, P < 0.001) and SCA subtypes (F = 2.636, P = 0.026, more improved SARA scores in SCA7 than in SCA6) were associated with better improvement in the SARA score (Table 2). In the spaghetti plot for 12 M SARA score changes, no significant difference in the baseline SARA score was observed between the responsive and nonresponsive groups (Fig. 3). The FARS II scores also significantly improved over time (F = 2.612, P = 0.038), and the FARS II scores at each evaluation time point (1 M, 3 M, 6 M, or 12 M) showed a significant improvement compared to baseline (P = 0.008, P = 0.006, P = 0.039, and P = 0.025, respectively) (Table 2 and Fig. 2B). Neither the FARS I score, nor the BI score exhibited a significant improvement compared to baseline (Supplemental Table 2Supplemental Fig. 2).

DEPs associated with the responsiveness to nilotinib

Serum proteomic analysis covering 895 proteins identified 50 DEPs, which were further classified into 3 classes as follows: class 1 (C > N > R, 22 DEPs), class 2 (R > C > N, 13 DEPs), and class 3 (R > N > C, 15 DEPs) (Fig. 4, list of DEPs in Supplemental Table 3, and full proteomic analysis data in Supplemental Dataset). The profiles of subjects and control subjects included in the proteomic analysis are summarized in Supplemental Table 4.

After Benjamini‒Hochberg adjustment for multiple comparisons, 4/50 (8.0%) proteins remained differentially expressed among the groups, including leucine-rich alpha-2-glycoprotein (LRG1) in class 1, vitamin-D binding protein (DBP) in class 2, and C4b-binding protein (C4BP) beta and alpha chain in class 3 (Table 3). A literature search revealed that all 4 proteins are involved in the autophagy process. Remarkably, DEPs with the lowest expression in the R group had a promotive effect on autophagy, whereas DEPs with the highest expression in the R group had an inhibitory effect on autophagy. LRG1 promotes autophagy via the regulation of the TGF-β-smad1/5 signaling pathway³²; DBP inhibits autophagy by sequestrating free vitamin D, which enhances autophagy via mTOR inhibition at the induction stage, Beclin1 and PI3K3 augmentation at the nucleation stage, and the activation of lysosomes at the maturation and degradation stage of autophagy^33–35; and C4BPs inhibit C3 and C4 binding, which activates the autophagy machinery^36,37; and inhibit protein S (PS) by binding to PS, blocking the site of PS responsible for the effect on the phagocytic index³⁸.