Intense Monitoring of JAK2V617F and Wild-Type Hematopoiesis during IFNα Exposure to Dissect Its Mechanism of Action

Interferon alfa (IFNα) is the only cytoreductive agent for myeloproliferative neoplasm (MPN) that can induce a deep molecular response by decreasing the variant allele frequency (VAF) of the MPN driver mutation JAK2V617F. However, not all MPN patients on IFNα enjoy a molecular response, and it commonly takes years of treatment to achieve. To identify early biomarkers that may predict a successful IFNα long-term response, we intensely monitored the JAK2V617F VAF in peripheral leukocytes of MPN patients during early IFNα treatment. We sought a feasible method for daily monitoring of JAK2V617F allele burden in study patients. We established that saliva is a faithful surrogate for peripheral blood (PB) to track somatic variants in hematopoietic cells. Saliva contains predominately white blood cells with some contaminating buccal epithelial cells; therefore, we developed a method using epigenetic cell counting to quantify buccal epithelial DNA to more accurately measure variants stemming from the hematopoietic cells in saliva samples (Soyfer et al, Blood Neoplasia 2024).

We have followed weekly saliva-derived JAK2V617F VAFs for a full year after starting IFNα in 12 patients (7 completed study, 5 on-study). The first 7 patients began with daily sampling, then relaxed to weekly sampling after 4 months once we determined there were no significant differences in predicted VAF trends between sampling schemes. There were two trends of VAF changes: 1) 8 patients had a gradual rise with a ~1.2x increase after 3 months, ~1.3x increase after 6 months, then plateaued for the remainder of the year; 2) 4 patients had a sharper rise to ~1.5x increase after 3 months, then plateaued for the remainder of the year. In both cases, blood count normalization didn’t begin until approximately 10 months.

For local patients, we also collected monthly PB samples, measured spleen length with ultrasound, and tracked MPN symptom burden. For 7 patients, spleen size transiently increased from normal (less than 12 cm) to slightly above normal (12-13 cm) after 2-4 months, with spleen size normalization occurring in the next 1-2 months.

We also explored the use of cell free DNA (cfDNA) from PB as a potential source of DNA that captures the mutational composition of bone marrow (BM). Monthly cfDNA samples were collected from plasma in a subset of 6patients, and they had a decrease in their cfDNA JAK2V617F VAF by 10%, demonstrating that the reduction of JAK2V617F allele burden is occurring earlier in cfDNA compared to PB.

In patients with known additional mutations, such as TET2 or DNMT3A, we are currently genotyping colonies to determine whether these variants are in the same or distinct clone as the JAK2V617F mutation, and to track how IFNα treatment impacts these clones.

In parallel to our human studies, we used MPN mouse models to intensely track the competition between JAK2V617F and WT stem/progenitor cells in the bone marrow following IFNα treatment. Mice transplanted with JAK2WT (CD45.1) and JAK2V617F (CD45.2) BM at a 50/50 ratio received daily subcutaneous injections of 20,000 IU mIFNα (Miltenyi) or PBS control. PB and BM aspirates (BMA) were collected at baseline and weekly or biweekly on therapy. We quantified JAK2V617F/WT chimerism in mature and progenitor populations in the BM. After only a few days of IFNα treatment, there was a 5% decrease of the %JAK2V617F in total cells as well as the lineage-negative population. In contrast, a transient rise of %JAK2V617F was seen in the Lineage-/cKit+ and Lineage-/cKit+/Sca-1+ populations. By 4 weeks, the %JAK2V617F to all 4 populations decreased to 20%, then plateaued. In contrast, PBS-treated mice had an increasing %JAK2V617F over time. Of PB leukocytes, the %JAK2V617F of total cells for IFNα-treated mice continued to drop well past 4 weeks.

Here, we used novel techniques to intensely track the competitive dynamics of JAK2V617F mutant versus wild-type cells in MPN patients and MPN mouse models during early IFNα treatment. Consistent with the model of IFNα preferentially inducing cycling in JAK2V617F HSC, we have found an early expansion of JAK2V617F mutant cells. With longer longitudinal assessment, we anticipate seeing continual reduction of JAK2V617F mutant cells in our MPN patients and mouse models.