Background/Objectives: Patients with chronic myeloid leukemia (CML) have a long life expectancy due to modern treatment. However, treatment may have adverse effects that hamper work ability. Methods: Patients aged 25–60 years diagnosed in 2002–2020 were included in this nationwide matched cohort study examining work ability from diagnosis (index date), including the need for permanent disability compensation (flexible job or disability pension). Each patient was matched 1:5 on sex, birth year, and level of comorbidity with citizens from the general Danish population without CML. The risks of requiring flexible job and disability pension were calculated by cause-specific hazard ratios (HRs) using Cox proportional hazards regression, and the Aalen–Johansen estimator was used to determine cumulative risks. Results: A total of 489 patients with CML and 2445 matched comparators were included. The median age was 46 years, and males comprised 59.5% of the cohort. Matched comparators were more likely to work at index date and 1, 3, 5, and 10 years after the index date (p < 0.001). The HRs of requiring both flexible job (HR 8.7 (95% confidence interval (CI): 6.1;12.2, p < 0.001)) and disability pension (HR 3.7 (95% CI: 2.8;4.9, p < 0.001)) were higher among patients diagnosed with CML compared to matched comparators. The cumulative risk of requiring flexible job and disability pension also increased in patients with CML compared to matched comparators (p < 0.001). Conclusions: Patients with CML have a reduced work ability compared to the general population. More research is needed to determine the cause of their loss of ability to work.

Prior studies combining pegylated interferon-α to Imatinib and Nilotinib in newly diagnosed CML patients have indicated an additive effect with faster and deeper molecular responses (SPIRIT, TIGER, NordCML002, NiloPEG). In this trial we used a bosutinib (BOS) backbone combined with low-dose ropeginterferon alfa-2b (BESREMi, AOP Health), which in contrast to previously tested interferons has a longer half-life and a more favorable tolerability profile. The aim was to investigate efficacy and tolerability of the combination, with the goal of deepening molecular response to eventually allow a higher proportion of patients to achieve treatment free remission. Patients were in 1st chronic phase and TKI-naïve. Inclusion criteria were standard including good organ function without history of liver, autoimmune or psychiatric disease. Based on clinical experience to improve tolerability, BOS was introduced at 200 mg OD and the dose titrated to 400 mg OD if tolerated. At month 3 (M3), patients who tolerated ≥300 mg OD and had transaminase levels grade ≤1 were randomized 1:1 for addition of ropeginterferon 50 μg every second week or not. The primary endpoint was MR4 at M12 in the intention-to-treat (IIT) population (all randomized patients). Other endpoints were safety and other response levels (e.g. MR4.5). Data up to M12 are presented. We also examined the response and tolerability of BOS up to M3 as the ramp-up dosing is not standard although frequently used in the clinic. 163 patients were included from 2019 to 2023 in 18 hospitals in Denmark (6), Finland (1), Norway (4) and Sweden (7). Despite gradual up-titration of BOS, many patients experienced toxicity as described in the BOS prescribing information, including grade 2, 3 and 4 transaminase elevations (11%; 16%; 3%). Dose reduction or interruption were insufficient to manage transaminase elevations, which frequently recurred upon BOS re-exposure. Compared with data from the BOS registration study (BFORE), GI AEs were less frequent with our present schedule. Twenty-seven % of patients could not be randomized: 15% for transaminitis, 4% for GI AEs and 8% for other reasons including inability to tolerate BOS 300mg OD. Non-randomized patients had M3 responses on par with randomized patients, indicating no harm of the dosing strategy despite the lower or intermittent BOS dose. Their responses at M6, M9 and M12 were similar to the standard arm. At M3, 118 patients were randomized, 58 to BOS and 60 to the combination. AEs were distributed evenly between study arms, but hematological toxicity (grades 3-4, 6.7% vs 0%), neuropsychiatric disorders (grade 2, 13% vs 4%) and infections grade 3-4 (13% vs 0%) were more frequent in the combination arm. Of note, transaminase elevations were similarly distributed, but analysis of attributability to the study drugs is ongoing and will be updated Overall, efficacy in the present study compared favorably with previous experience with BOS. The combination arm demonstrated a clear trend for faster and deeper response at all time points: The combination arm compared with the standard arm showed MR4 or better in 22.0% vs 13.8% at M6, 32.8% vs 17.2% at M9, and 38.3% vs 31.0% at M12. Attainment of MR4.5 or better was at M6, M9 and M12, 11% vs 5,2%, 22,4% vs 6,9% and 31,7% vs 20,7%, respectively. Discontinuation of treatment occurred in 22% of patients in the combination arm and 8,3% in the standard arm, of whom 11,5% and 5% for treatment resistance. The primary endpoint in the IIT dataset was not statistically significantly different between treatment arms; note that all patients were included in the ITT analysis even if no interferon was administered or the TKI was switched. Most switched patients received 1st and 2nd generation TKIs, but some were transplanted or received ponatinib. A per protocol analysis and data on drug exposure will be presented at the meeting. The step-up dosing strategy for BOS reduced GI toxicity, but not transaminitis. Efficacy was excellent in comparison with the registration study (BFORE). The combination of ropeginterferon and BOS was safe and efficacious. Responses occurred earlier and were deeper with the combination.

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.

Objectives Treatment-free remission (TFR) has emerged as a treatment goal in chronic myeloid leukemia in the chronic phase (CML-CP). Attempts to increase proportion of patients achieving TFR include combination of tyrosine kinase inhibitors (TKI) and other drugs. Interferon-alpha in addition to TKI has shown promising efficacy but with dose-dependent toxicity and discontinuations. NordCML007 was initiated to study the efficacy and safety of low dose pegylated IFN-alpha (PegIFN-alpha) in combination with dasatinib (DAS) in CML-CP. Methods Forty patients with newly diagnosed CML-CP were given DAS upfront. After month 3 (M3) 15 mu g/wk of PegIFN-alpha was added and increased to 25 mu g/wk from M7 until M15. DAS treatment was continued and adverse events and BCR-ABL1 qRT-PCR values were reported yearly after M24. Results from M1 to M18 have previously been published, and here we present long-term data. Results After 5 years of follow-up, there were no suspected unexpected serious adverse reactions, no increase in serosal effusions, no disease progressions and no CML-related deaths. Rates of MR3.0 (MMR), MR4.0 and MR4.5 were 84.6%, 64.1% and 51.3% respectively at M60, and 95% of patients reached MMR at some point during the study. Conclusion Initial addition of PegIFN-alpha to DAS shows good long-term efficacy without increased toxicity.

Changes in the immune system induced by tyrosine kinase inhibitors (TKI) have been shown to positively correlate with therapy responses in chronic myeloid leukemia (CML). However, only a few longitudinal studies exist and no randomized comparisons between two TKIs have been reported. Therefore, we prospectively analyzed the immune system of newly diagnosed CML patients treated with imatinib (n = 20) or bosutinib (n = 13), that participated in the randomized BFORE trial (NCT02130557). Comprehensive immunophenotyping, plasma protein profiling, and functional assays to determine activation levels of T and NK cells were performed at diagnosis, 3, and 12 months after therapy start. All results were correlated with clinical parameters such as Sokal risk and BCR-ABL load measured according to IS%. At diagnosis, low Sokal risk CML patients had a higher frequency of cytotoxic cells (CD8 + T and NK cells), increased cytotoxic potential of NK cells and lower frequency of naïve and central memory CD4 + T cells. Further, soluble plasma protein profile divided patients into two distinct clusters with different disease burden at diagnosis. During treatment, BCR-ABL IS% correlated with immunological parameters such as plasma proteins, together with different memory subsets of CD4+ and CD8 + T cells. Interestingly, the proportion and cytotoxic potential of NK cells together with several soluble proteins increased during imatinib treatment. In contrast, no major immunological changes were observed during bosutinib treatment. In conclusion, imatinib and bosutinib were shown to have differential effects on the immune system in this randomized clinical trial. Increased number and function of NK cells were especially observed during imatinib therapy.

Objective Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). The assay amplifies both e13a2 and e14a2 BCR-ABL1 transcript variants. Observing distinct variant-dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR-ABL1. Methods We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital [ZUH], Aarhus University Hospital [AU], and Rigshospitalet [RH]) on cell lines expressing either the e13a2 or e14a2 BCR-ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR). Results Although no significant differences were found in amplification efficiencies of the transcript variants, Bland-Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6-, 6.5-, and 1.8-fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7-fold difference between the e13a2 and e14a2 variants. Conclusion Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.

Dasatinib (DAS) and interferon-α have antileukemic and immunostimulatory effects and induce deep responses in chronic myeloid leukemia (CML). We assigned 40 newly diagnosed chronic-phase CML patients to receive DAS 100 mg o.d. followed by addition of pegylated interferon-α2b (PegIFN) after 3 months (M3). The starting dose of PegIFN was 15 μg/week and it increased to 25 μg/week at M6 until M15. The combination was well tolerated with manageable toxicity. Of the patients, 84% remained on PegIFN at M12 and 91% (DAS) and 73% (PegIFN) of assigned dose was given. Only one patient had a pleural effusion during first year, and three more during the second year. After introduction of PegIFN we observed a steep increase in response rates. Major molecular response was achieved in 10%, 57%, 84% and 89% of patients at M3, M6, M12 and M18, respectively. At M12, MR4 was achieved by 46% and MR4.5 by 27% of patients. No patients progressed to advanced phase. In conclusion, the combination treatment appeared safe with very promising efficacy. A randomized comparison of DAS±PegIFN is warranted.

Background: Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) are not entirely selective for the BCR-ABL1 kinase but also inhibit a variety of other kinases, sometimes triggering unpredicted biological effects. As an example, the TKIs dasatinib and bosutinib both inhibit Src-kinases, which are important mediators of T-cell function. Earlier in vitro data has shown that dasatinib can suppress activation and proliferation of T and NK cells, but it can also elicit signs of immunostimulation in patients, including rapid mobilization of lymphocytes and LGL lymphocytosis. No extensive analyses of the immunological in vivoeffects of bosutinib have been performed thus far. Therefore, we aimed at characterizing T and NK cell phenotypes and functional features in CML patients in a clinical setting in the context of first-line bosutinib and imatinib treatment.

Background: In chronic myeloid leukemia (CML) the combination treatment of tyrosine kinase inhibitors (TKIs) with interferon-α (IFN-α) has proved to be effective and well-tolerated. IFN-α has long-term immunomodulatory effects, and when combined to TKI therapy, it may increase the success rates for treatment free remission. In our recent clinical trial NordCML007, a low-dose pegylated IFN-α was combined with dasatinib therapy. As dasatinib is also known to have immunostimulatory effects (activation of T and NK cells and downregulation of regulatory T cells), we aimed to monitor the immune effects of dasatinib and IFN-α combination treatment.

Aim: The Danish National Chronic Myeloid Neoplasia Registry (DCMR) is a population-based clinical quality database, introduced to evaluate diagnosis and treatment of patients with chronic myeloid malignancies. The aim is to monitor the clinical quality at the national, regional, and hospital departmental levels and serve as a platform for research.Study population: The DCMR has nationwide coverage and contains information on patients diagnosed at hematology departments from January 2010 onward, including patients with essential thrombocythemia, polycythemia vera, myelofibrosis, unclassifiable myeloproliferative neoplasms, chronic myelomonocytic leukemia, and chronic myeloid leukemia.Main variables: Data are collected using standardized registration forms (so far up to four forms per patient), which are consecutively filled out online at time of diagnosis, after 2-year and 5-year follow-ups, and at end of follow-up. The forms include variables that describe clinical/paraclinical assessments, treatment, disease progression, and survival - disease-specific variables - as well as variables that are identical for all chronic myeloid malignancies.Descriptive data: By the end of 2014, the DCMR contained data on 2,690 patients with an inclusion rate of similar to 500 patients each year. Since the registry was established, annual reports have shown consistently high national coverage and data completeness, >= 90% and >= 88%, respectively.Conclusion: The DCMR is a national database used for monitoring the quality of patient care in patients with chronic myeloid malignancies, but until validation has been conducted, the data must be used with caution. However, the DCMR is a valuable data source accessible to clinicians and researchers.

The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its passage from blood to tissues. Nitrogen cavitation was developed as a method for disruption of cells on the assumption that sudden reduction of the partial pressure of nitrogen would lead to aeration of nitrogen dissolved in the lipid bilayer of plasma membranes. We find that cells are broken by the shear stress that is associated with passage through the outlet valve under high pressure and that this results in disruption of the neutrophil cell membrane while granules remain intact. The unique properties of Percoll as a sedimentable density medium with no inherent tonicity or viscosity are used for creation of continuous density gradients with shoulders in the density profile created to optimize the physical separation of granule subsets and light membranes. Immunological methods (sandwich enzyme-linked immunosorbent assays) are used for quantitation of proteins that are characteristic constituents of the granule subsets of neutrophils.

BACKGROUND:β-Microseminoprotein (MSMB) is an abundant protein in seminal plasma. Most of it is present as a free protein but a small part is bound to cysteine-rich secretory protein 3 (CRISP3) as a non-covalent complex. Even though their physiological function is unknown, both MSMB and CRISP3 have been ascribed roles in prostate carcinogenesis. Thus, several recent experimental studies indicate a tumor-suppressor role for MSMB. The present study was undertaken in order to evaluate, for the first time, the expression of MSMB and CRISP3 in ovaries and in ovarian tumors and to determine if their expression might indicate a role in ovarian tumor development.MATERIALS AND METHODS:Biopsies from prospectively collected samples from ovaries and benign, borderline and invasive ovarian tumors were analyzed for expression of MSMB and CRISP3 by immunohistochemistry. In patients with ovarian cancer the expression was compared to survival.RESULTS:Both MSMB and CRISP3 were strongly stained in ovarian epithelial cells and weakly stained in the stroma. In ovarian blood vessels, CRISP3 exhibited strong to medium staining, while MSMB was only weakly expressed. In benign and borderline tumors the staining pattern was similar to the one observed in the ovaries. In invasive neoplasms, the expression of MSMB in the tumor cells was significantly reduced. In univariate analysis, decreased expression of MSMB correlated to reduced survival. No correlation was found with stage, the strongest prognostic indicator for ovarian cancer, which supports an independent role of MSMB in ovarian carcinogenesis. For CRISP3, a staining pattern comparable to that for MSMB was observed in all groups, except the fact that decreased expression was not observed in invasive tumor cells.CONCLUSION:MSMB and CRISP3 were widely distributed in ovaries and in ovarian tumors; the expression of MSMB fits well with a tumor-suppressor function in ovarian carcinogenesis.

Background: CRISP-3 was previously shown to be bound to alpha B-1-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments.Methods: We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing.Results: We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen I in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG.General significance: It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by AIBG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. (c) 2010 Elsevier B.V. All rights reserved.