Neutrophil-to-lymphocyte ratio and all-cause mortality with and without myeloproliferative neoplasms-a Danish longitudinal study.

The neutrophil-to-lymphocyte ratio(NLR) is increased in chronic inflammation and myeloproliferative neoplasms (MPN). We hypothesize that NLR is associated with all-cause mortality and mortality by comorbidity burden in the general population and individuals with MPN. We included 835,430 individuals from The Danish General Suburban Population Study, general practitioners, and outpatient clinics. We investigated NLR on mortality stratified by prevalent and incident MPN, essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), comorbidity burden (CCI-score), and the Triple-A risk score using hazard ratio (HR) and 95% confidence interval (95%CI). NLR 1-1.9 was the reference level. During a median follow-up of 11.2 years, 197,802 deaths were recorded. All-cause mortality increased for a stepwise increasing NLR with a HR (95%CI) for NLR ≥ 6 of 2.06(2.03-2.09) for the whole population and 2.93(2.44-3.50) in prevalent MPN. ET, PV, and MF had a HR (95%CI) for NLR ≥ 2 of 2.14(1.71-2.69), 2.19(1.89-2.54), and 2.31(1.91-2.80). Results were similar for incident MPN. Mortality was higher for stepwise increasing NLR and CCI-score(pinteraction < 2×10-16), with a HR for NLR ≥ 6 of 2.23(2.17-2.29), 4.10(4.01-4.20), and 7.69(7.50-7.89), for CCI-score 0, 1-2, or ≥3. The Triple-A risk score demonstrated alignment with NLR. Increasing NLR and comorbidity burden were associated with lower survival in individuals without MPN but were even worse in prevalent and incident MPN, ET, PV, and MF.

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.

The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells.

CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.