Dynamics of Calrvariant Allele Frequency with Therapy in Myeloproliferative Neoplasms
Authors: Ng Zi Yun , Cottin Laurane , Luque Paz Damien , Orvain Corentin , Laribi Kamel , Ianotto Jean-Christophe , Lippert Eric , Khan Alesia , Lock Daniel , Ugo Valerie , Harrison Claire , O'Sullivan Jennifer 
Published: December 2024

Introduction:
Sequential JAK2V617F variant allele frequency (VAF) monitoring in treated myeloproliferative neoplasm (MPN) patients has shown molecular responses (MR) which correlate with improved clinical outcomes. However, the implications of serial CALR VAF testing are less certain. Pegylated interferon-alpha (IFN) has been shown to variably reduce CALRVAF but change with other treatments such as hydroxycarbamide (HC), anagrelide (ANA) and ruxolitinib (RUX), a JAK inhibitor, is less well described.
Methods:
Clinical and molecular information was collated from clinical trial and unpublished datasets across UK and French centres. CALR VAF was quantified using next-generation sequencing or fragment analysis approaches at the respective centres. MR were defined as per the IWG-MRT/ELN 2013 criteria, where complete molecular response (CMR) was defined as eradication of a preexisting abnormality and a partial molecular response (PMR) as ≥50% decrease in VAF. An exploratory endpoint, “PMR20”, was included, defined as 20-49% decrease in VAF. PMR20 was chosen a priori since it has been shown to correlate with clinical/histological response and overall survival in clinical trials (Mascarenhas et al, JCO 2021, Pemmaraju et al, JCO 2023). Otherwise, VAF changes 0-1% were categorised as “no change”, a decrease in VAF between 2-19% as “non-MR decrease” and an increase of ≥2% as “increase”.
Results:
Serial testing (2 timepoints separated by a minimum of 6 months) was available in 131 patients. MPN subtypes in the cohort included essential thrombocythemia [ET] (n=74, 56.1%), pre-fibrotic myelofibrosis [pre-PMF] (n=13, 9.9%), primary myelofibrosis [PMF] (n=32, 24.2%), post-ET myelofibrosis [PETMF] (n=9, 6.8%), MPN-unclassified [MPN-U (n=3, 2.2%) and blast-phase MPN (n=1, 0.8%). Median time to second sampling was 4.1 years (0.5 - 18.5 years). The patients were treated with the following: ANA (n=14, 11.6%), HC (n=40, 33.3%), RUX (n=33, 27.5%), IFN (n=33, 27.5%) and 12 had other treatments. The median duration of treatment was 41 months (range 1-175 months).
Overall MR consisted of CMR (n=2, 1.5%), PMR (n=10, 7.6%) and PMR20 (n=23, 17.6%). Non-MR decreases, no changes and increases occurred in 13% (n=17), 12.2% (n=16) and 48.1% (n=63) respectively. Of those with extended mutational testing (n=47), 31.8% had an additional mutation. The presence of additional mutations had no impact on molecular response (p=0.6). MR occurred from year 1 to beyond 5 years and no correlation with VAF timing and MR was observed.
There was a significant association between MR and treatment groups (p=0.004). As expected, IFN-treated patients had the highest frequency overall of molecular responses (n=13, 39.4%); PMR: n=7, 21.2% and PMR20: n=6, 18.2%. PMR20 was most frequent in RUX-treated patients (n=7 [5 PMF, 2 ET], 21.1%) but fewer had PMR (n=1, 3.2%). CMR occurred in 6.1% (n=2, RUX-treated). HC-treated patients also experienced MR with PMR20 in 17.5% (n=7) and but few PMR (n=2, 5%). ANA-treated patients, in contrast, had few MR (PMR20 in 7.1% (n=1)). Non-MR decreases were most often seen in RUX-treated (n=5), followed by IFN (n=4), HC (n=4) and one ANA-treated patient. The majority of ANA-treated patients had an increase in VAF (n=12, 85.7%). Increase in VAF was observed in 52.5%, 45.5% and 27.2% of HC-, IFN- and RUX-treated patients, respectively.
As per IWG/ELN 2013 criteria, patients with PMR most often had a complete response (CR), (n=9, 90%, p=0.006). Patients with no clinical response more often had an increase (n=11, 64.7%) or no change (n=4, 23.5%) in VAF and few MR (n=2, 11.8%). There was no significant association between MR and gender (p=0.06), MPN subtype (p=0.36), prior number of treatment lines (p=0.06), or age at time of VAF (p=0.3).
Conclusion:
In summary, in this largest series of patients to date assessing MR to treatment in CALR mutated MPN, we report that MR are mainly PMR / PMR20 and can occur early from 1-2 years. MR occurred across treatments including IFN as reported previously but in line with the MAJIC PV study (Harrison et al, JCO 2023), we observed MR in almost a third of RUX-treated patients inclusive of ET and MF. Intriguingly, majority of ANA-treated patients had an increase in CALR VAF. Whether increasing VAF is a concern is also unclear but the correlation between anagrelide and risk of PETMF and the current association with increased VAF is potentially relevant in this context.