Mathematical modelling of stem and progenitor cell dynamics during ruxolitinib treatment of patients with myeloproliferative neoplasms.

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms are a group of slowly progressing haematological malignancies primarily characterised by an overproduction of myeloid blood cells. Patients are treated with various drugs, including the JAK1/2 inhibitor ruxolitinib. Mathematical modelling can help propose and test hypotheses of how the treatment works. Materials and methods: We present an extension of the Cancitis model, which describes the development of myeloproliferative neoplasms and their interactions with inflammation, that explicitly models progenitor cells and can account for treatment with ruxolitinib through effects on the malignant stem cell response to cytokine signalling and the death rate of malignant progenitor cells. The model has been fitted to individual patients' data for the JAK2 V617F variant allele frequency from the COMFORT-II and RESPONSE studies for patients who had substantial reductions (20 percentage points or 90% of the baseline value) in their JAK2 V617F variant allele frequency (n = 24 in total). Results: The model fits very well to the patient data with an average root mean square error of 0.0249 (2.49%) when allowing ruxolitinib treatment to affect both malignant stem and progenitor cells. This average root mean square error is much lower than if allowing ruxolitinib treatment to affect only malignant stem or only malignant progenitor cells (average root mean square errors of 0.138 (13.8%) and 0.0874 (8.74%), respectively). Discussion: Systematic simulation studies and fitting of the model to the patient data suggest that an initial reduction of the malignant cell burden followed by a monotonic increase can be recapitulated by the model assuming that ruxolitinib affects only the death rate of malignant progenitor cells. For patients exhibiting a long-term reduction of the malignant cells, the model predicts that ruxolitinib also affects stem cell parameters, such as the malignant stem cells' response to cytokine signalling.

Case Report: First longitudinal study of a patient with CALR positive clonal hematopoiesis of indeterminate potential developing into pre-fibrotic myelofibrosis.

Initial diagnosis of overt myeloproliferative neoplasms (MPNs) represents the juncture during clonal evolution when symptoms or complications prompt an afflicted individual to seek medical attention. In 30-40% of the MPN subgroups essential thrombocythemia (ET) and myelofibrosis (MF), somatic mutations in the calreticulin gene (CALR) are drivers of the disease resulting in constitutive activation of the thrombopoietin receptor (MPL). In the current study, we describe a healthy CALR mutated individual during a 12 year follow-up from initial identification of CALR clonal hematopoiesis of indeterminate potential (CHIP) to the diagnosis of pre-MF. The pre-diagnostic exponential development dynamics of the malignant clone demonstrated close correlation with the platelet counts, neutrophil-to-lymphocyte (NLR) ratio, and inversely correlated to hemoglobin and erythrocyte counts. Backward extrapolation of the growth rate indicated the potential for discovery of the malignant clone many years prior to presentation of overt disease, opening a window of opportunity for early treatment intervention. We did not find any additional mutations associated with MPNs and the current case report provides novel information regarding the development of a driver mutation and the association with blood cell counts prior to clinical manifestation of symptoms suggesting that pre-diagnostic dynamics may supplement future diagnostic criteria for early diagnosis and intervention in MPN patients.

Interferon alpha-2 treatment reduces circulating neutrophil extracellular trap levels in myeloproliferative neoplasms.

Neutrophil extracellular traps (NETs) may play a pathogenic role in the thrombosis associated with myeloproliferative neoplasms (MPNs). We measured serum NET levels in 128 pretreatment samples from patients with MPNs and in 85 samples taken after 12 months of treatment with interferon alpha-2 (PEG-IFNα-2) formulations or hydroxyurea (HU). No differences in NET levels were observed across subdiagnoses or phenotypic driver mutations. In PV, a JAK2V617F+ allele burden ≥50% associated with increased NET levels (p = 0.006). Baseline NET levels correlated with neutrophil count (r = 0.29, p = 0.001), neutrophil-to-lymphocyte ratio (r = 0.26, p = 0.004) and JAK2V617F allele burden (r = 0.22, p = 0.03), particularly in patients with PV and with allele burden ≥50% (r = 0.50, p = 0.01, r = 0.56, p = 0.002 and r = 0.45, p = 0.03 respectively). In PV, after 12 months of treatment, NET levels decreased on average by 60% in patients with allele burden ≥50%, compared to only 36% in patients with an allele burden <50%. Overall, treatment with PEG-IFNα-2a or PEG-IFNα-2b reduced NETs levels in 77% and 73% of patients, respectively, versus only 53% of HU-treated patients (average decrease across treatments: 48%). Normalization of blood counts did not per se account for these reductions. In conclusion, baseline NET levels correlated with neutrophil count, NLR and JAK2V617F allele burden, and IFNα was more effective at reducing prothrombotic NET levels than HU.

A novel integrated biomarker index for the assessment of hematological responses in MPNs during treatment with hydroxyurea and interferon-alpha2.

BACKGROUND: Conventional cytoreductive therapy for patients with chronic Philadelphia-negative myeloproliferative neoplasms (MPNs) includes hydroxyurea (HU), interferon-alpha2 (IFN), and anagrelide. HU is worldwide the most used cytoreductive agent, which lowers elevated blood cell counts within days in the large majority of patients. However, some patients may experience rebound cytosis when HU is reduced due to cytopenia, thereby potentially giving rise to fluctuating cell counts during therapy. Such rapid oscillations may be harmful and potentially elicit thrombosis. Treatment with IFN gradually lowers elevated cell counts within weeks and when the dosage is reduced, the cell counts do not rapidly increase but are sustained within the normal range in the large majority of patients. Conventional hematological response criteria are among others based upon single absolute cell count values and do not take into account the relative decreases toward normal for each cell count. MATERIALS, METHODS & RESULTS: Using serial data from the Danish DALIAH trial, we herein describe a novel integrated biomarker index for the assessment of hematological and molecular (JAK2V617F) responses in patients with MPNs during treatment with IFN or HU. DISCUSSION: This novel tool convincingly displays the superiority of IFN versus HU in normalizing elevated cell counts. Our results need to be validated in larger studies but already now call for studies of the safety and efficacy of combination therapy during the initial treatment of patients with MPNs.

HSC Niche Dynamics in Regeneration, Pre-malignancy, and Cancer: Insights From Mathematical Modeling.

The hematopoietic stem cell (HSC) niche is a crucial driver of regeneration and malignancy. Its interaction with hematopoietic and malignant stem cells is highly complex and direct experimental observations are challenging. We here develop a mathematical model which helps relate processes in the niche to measurable changes of stem and non-stem cell counts. HSC attached to the niche are assumed to be quiescent. After detachment HSC become activated and divide or differentiate. To maintain their stemness, the progeny originating from division must reattach to the niche. We use mouse data from literature to parametrize the model. By combining mathematical analysis and computer simulations, we systematically investigate the impact of stem cell proliferation, differentiation, niche attachment, and detachment on clinically relevant scenarios. These include bone marrow transplantation, clonal competition, and eradication of malignant cells. According to our model, sampling of blood or bulk marrow provides only limited information about cellular interactions in the niche and the clonal composition of the stem cell population. Furthermore, we investigate how interference with processes in the stem cell niche could help to increase the effect of low-dose chemotherapy or to improve the homing of genetically engineered cells.

Clonal haematopoiesis of indeterminate potential and impaired kidney function-A Danish general population study with 11 years follow-up.

The myeloproliferative neoplasms are associated with chronic kidney disease but whether clonal haematopoiesis of indeterminate potential (CHIP) is associated with impaired kidney function is unknown. In the Danish General Suburban Population Study (N = 19 958) from 2010 to 2013, 645 individuals were positive for JAK2V617F (N = 613) or CALR (N = 32) mutations. Mutation-positive individuals without haematological malignancy were defined as having CHIP (N = 629). We used multiple and inverse probability weighted (IPW)-adjusted linear regression analysis to estimate adjusted mean (95% confidence interval) differences in estimated glomerular filtration rate (eGFR; ml/min/1.73 m2 ) by mutation status, variant allele frequency (VAF%), blood cell counts, and neutrophil-to-lymphocyte ratio (NLR). We performed 11-year longitudinal follow-up of eGFR in all individuals. Compared to CHIP-negative individuals, the mean differences in eGFR were -5.6 (-10.3, -0.8, p = .02) for CALR, -11.9 (-21.4, -2.4, p = 0.01) for CALR type 2, and -10.1 (-18.1, -2.2, p = .01) for CALR with VAF ≥ 1%. The IPW-adjusted linear regression analyses showed similar results. NLR was negatively associated with eGFR. Individuals with CALR type 2 had a worse 11-year longitudinal follow-up on eGFR compared to CHIP-negative individuals (p = .004). In conclusion, individuals with CALR mutations, especially CALR type 2, had impaired kidney function compared to CHIP-negative individuals as measured by a lower eGFR at baseline and during 11-year follow-up.

Interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms favorably impacts deregulation of oxidative stress genes and antioxidative defense mechanisms.

Chronic inflammation is considered a major driving force for clonal expansion and evolution in the Philadelphia-negative myeloproliferative neoplasms, which include essential thrombocythemia, polycythemia vera and primary myelofibrosis (MPNs). One of the key mutation drivers is the JAK2V617F mutation, which has been shown to induce the generation of reactive oxygen species (ROS). Using whole blood gene expression profiling, deregulation of several oxidative stress and anti-oxidative defense genes has been identified in MPNs, including significant downregulation of TP53, the NFE2L2 or NRF2 genes. These genes have a major role for maintaining genomic stability, regulation of the oxidative stress response and in modulating migration or retention of hematopoietic stem cells. Therefore, their deregulation might give rise to increasing genomic instability, increased chronic inflammation and disease progression with egress of hematopoietic stem cells from the bone marrow to seed in the spleen, liver and elsewhere. Interferon-alpha2 (rIFNα) is increasingly being recognized as the drug of choice for the treatment of patients with MPNs. Herein, we report the first gene expression profiling study on the impact of rIFNα upon oxidative stress and antioxidative defense genes in patients with MPNs (n = 33), showing that rIFNα downregulates several upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes. Treatment with rIFNα induced upregulation of 19 genes in ET and 29 genes in PV including CXCR4 and TP53. In conclusion, this rIFNα- mediated dampening of genotoxic damage to hematopoietic cells may ultimately diminish the risk of additional mutations and accordingly clonal evolution and disease progression towards myelofibrotic and leukemic transformation.