CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors.

Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

Patient-reported symptom monitoring and adherence to therapy in patients with newly diagnosed chronic myeloid leukemia.

BACKGROUND: The authors assessed the clinical utility of patient-reported symptom monitoring in the setting of newly diagnosed chronic myeloid leukemia (CML). The primary objective was to evaluate adherence to therapy. METHODS: The authors conducted an international prospective study that included patients with newly diagnosed, chronic-phase CML. Before clinical consultation, patients were provided a tablet computer to self-rate their symptoms, and the results were available in real time to each physician during the patient's visit. Adherence was assessed by pill count and with a validated self-reported questionnaire. The proportions of optimal responders at 3 and 6 months were assessed according to the European LeukemiaNet criteria. RESULTS: Between July 2020 and August 2021, 94 patients with a median age of 57 years were enrolled. Pill count adherence analysis indicated that 86 of 93 evaluable patients (92.5%) took at least 90% of prescribed tyrosine kinase inhibitor therapy during the 6-month observation period. The online platform was well accepted by patients and physicians. An optimal response was achieved by 69 of 79 patients (87.3%) at 3 months and by 61 of 81 patients (75.3%) at 6 months. CONCLUSIONS: Patient-reported symptom monitoring from the beginning of therapy in patients with CML may be critical to improve adherence to therapy and early molecular response rates (ClinicalTrials.gov identifier NCT04384848).

Incidence of blast phase in myelofibrosis patients according to anemia severity at ruxolitinib start and during therapy.

BACKGROUND: Anemia is frequently present in patients with myelofibrosis (MF), and it may be exacerbated by treatment with the JAK2-inhibitor ruxolitinib (RUX). Recently, a relevant blast phase (BP) incidence has been reported in anemic MF patients unexposed to RUX. METHODS: The authors investigated the incidence of BP in 886 RUX-treated MF patients, included in the "RUX-MF" retrospective study. RESULTS: The BP incidence rate ratio (IRR) was 3.74 per 100 patient-years (3.74 %p-y). At therapy start, Common Terminology Criteria for Adverse Events grade 3-4 anemia (hemoglobin [Hb] <8 g/dL) and severe sex/severity-adjusted anemia (Hb <8/<9 g/dL in women/men) were present in 22.5% and 25% patients, respectively. IRR of BP was 2.34 in patients with no baseline anemia and reached respectively 4.22, 4.89, and 4.93 %p-y in patients with grade 1, 2, and 3-4 anemia. Considering the sex/severity-adjusted Hb thresholds, IRR of BP was 2.85, 4.97, and 4.89 %p-y in patients with mild/no anemia, moderate, and severe anemia. Transfusion-dependent patients had the highest IRR (5.03 %p-y). Progression-free survival at 5 years was 70%, 52%, 43%, and 27% in patients with no, grade 1, 2, and 3-4 anemia, respectively (p < .001). At 6 months, 260 of 289 patients with no baseline anemia were receiving ruxolitinib, and 9.2% had developed a grade 3-4 anemia. By 6-month landmark analysis, BP-free survival was significantly worse in patients acquiring grade 3-4 anemia (69.3% vs. 88.1% at 5 years, p < .001). CONCLUSIONS: This study highlights that anemia correlates with an increased risk of evolution into BP, both when present at baseline and when acquired during RUX monotherapy. Innovative anemia therapies and disease-modifying agents are warranted in these patients.

The new Systematic Coronary Risk Evaluation (SCORE2 and SCORE2-OP) estimates the risk of arterial occlusive events in chronic myeloid leukemia patients treated with nilotinib or ponatinib.

Patients with chronic myeloid leukemia (CML) treated with nilotinib or ponatinib may experience arterial occlusive events (AOEs). It is currently recommended to thoroughly assess cardiovascular risk factors before treating CML. We identified 455 consecutive CML adult patients, 335 treated with nilotinib and 120 with ponatinib; 380 patients without previous cardiovascular diseases or diabetes were stratified according to the Systematic Coronary Risk Evaluation (SCORE2) and SCORE2-Older Persons (SCORE2-OP). This updated algorithm from the European Society of Cardiology (ESC) estimates a 10-year risk of fatal and non-fatal cardiovascular diseases. It is based on sex, age, smoking habits, systolic blood pressure, non-high-density lipoprotein cholesterol, and European geographical region of cardiovascular risk. The SCORE2/SCORE2-OP algorithm translated more patients (50.2%) to the high-very high cardiovascular risk category than the previous SCORE (25.3%). Patients with a high to very high SCORE2/SCORE2-OP risk showed a significantly higher incidence rate of AOEs (69.2% vs. 46.5%, p < 0.001). The older SCORE was less specific in estimating AOEs in patients classified as low-intermediate risk (69.8 vs. 54.2%). In multivariate analysis, no associations were found between AOEs and gender, age, and type or dose of tyrosine kinase inhibitor. Only the SCORE2/SCORE2-OP risk was confirmed as a significant predictive factor (p = 0.028; hazard ratio = 2.2; 95% confidence interval = 1.1-4.5). Patients with AOEs required, in most cases, imaging diagnostic tests, additional drugs, and sometimes invasive procedures, increasing access to visits and hospital management. This real-life study suggested that the SCORE2 and SCORE2-OP charts could help identify cardiovascular fragility in CML patients providing them with more attention and a proper TKI selection.

Ropeginterferon phase 2 randomized study in low-risk polycythemia vera: 5-year drug survival and efficacy outcomes.

In patients with low-risk polycythemia vera, exposure to low-dose Ropeginterferon alfa-2b (Ropeg) 100 µg every 2 weeks for 2 years was more effective than the standard treatment of therapeutic phlebotomy in maintaining target hematocrit (HCT) (< 45%) with a reduction in the need for phlebotomy without disease progression. In the present paper, we analyzed drug survival, defined as a surrogate measure of the efficacy, safety, adherence, and tolerability of Ropeg in patients followed up to 5 years. During the first 2 years, Ropeg and phlebotomy-only (Phl-O) were discontinued in 33% and 70% of patients, respectively, for lack of response (12 in the Ropeg arm vs. 34 in the Phl-O arm) or adverse events (6 vs. 0) and withdrawal of consent in (3 vs. 10). Thirty-six Ropeg responders continued the drug for up to 3 years, and the probability of drug survival after a median of 3.15 years was 59%. Notably, the primary composite endpoint was maintained in 97%, 94%, and 94% of patients still on drug at 3, 4, and 5 years, respectively, and 60% of cases were phlebotomy-free. Twenty-three of 63 Phl-O patients (37%) failed the primary endpoint and were crossed over to Ropeg; among the risk factors for this failure, the need for more than three bloodletting procedures in the first 6 months emerged as the most important determinant. In conclusion, to improve the effectiveness of Ropeg, we suggest increasing the dose and using it earlier driven by high phlebotomy need in the first 6 months post-diagnosis.

Ruxolitinib in cytopenic myelofibrosis: Response, toxicity, drug discontinuation, and outcome.

BACKGROUND: Patients with cytopenic myelofibrosis (MF) have more limited therapeutic options and poorer prognoses compared with patients with the myeloproliferative phenotype. AIMS AND METHODS: Prognostic correlates of cytopenic phenotype were explored in 886 ruxolitinib-treated patients with primary/secondary MF (PMF/SMF) included in the RUX-MF retrospective study. Cytopenia was defined as: leukocyte count <4 × 109 /L and/or hemoglobin <11/<10 g/dL (males/females) and/or platelets <100 × 109 /L. RESULTS: Overall, 407 (45.9%) patients had a cytopenic MF, including 249 (52.4%) with PMF. In multivariable analysis, high molecular risk mutations (p = .04), intermediate 2/high Dynamic International Prognostic Score System (p < .001) and intermediate 2/high Myelofibrosis Secondary to Polycythemia Vera and Essential Thrombocythemia Prognostic Model (p < .001) remained associated with cytopenic MF in the overall cohort, PMF, and SMF, respectively. Patients with cytopenia received lower average ruxolitinib at the starting (25.2 mg/day vs. 30.2 mg/day, p < .001) and overall doses (23.6 mg/day vs. 26.8 mg/day, p < .001) and achieved lower rates of spleen (26.5% vs. 34.1%, p = .04) and symptom (59.8% vs. 68.8%, p = .008) responses at 6 months compared with patients with the proliferative phenotype. Patients with cytopenia also had higher rates of thrombocytopenia at 3 months (31.1% vs. 18.8%, p < .001) but lower rates of anemia (65.6% vs. 57.7%, p = .02 at 3 months and 56.6% vs. 23.9% at 6 months, p < .001). After competing risk analysis, the cumulative incidence of ruxolitinib discontinuation at 5 years was 57% and 38% in patients with cytopenia and the proliferative phenotype (p < .001), whereas cumulative incidence of leukemic transformation was similar (p = .06). In Cox regression analysis adjusted for Dynamic International Prognostic Score System score, survival was significantly shorter in patients with cytopenia (p < .001). CONCLUSIONS: Cytopenic MF has a lower probability of therapeutic success with ruxolitinib as monotherapy and worse outcome. These patients should be considered for alternative therapeutic strategies.

Determinants of frontline tyrosine kinase inhibitor choice for patients with chronic-phase chronic myeloid leukemia: A study from the Registro Italiano LMC and Campus CML.

BACKGROUND: Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) approved in Italy for frontline treatment of chronic-phase chronic myeloid leukemia (CP-CML). The choice of TKI is based on a combined evaluation of the patient's and the disease characteristics. The aim of this study was to analyze the use of frontline TKI therapy in an unselected cohort of Italian patients with CP-CML to correlate the choice with the patient's features. METHODS: A total of 1967 patients with CP-CML diagnosed between 2012 and 2019 at 36 centers throughout Italy were retrospectively evaluated; 1089 patients (55.4%) received imatinib and 878 patients (44.6%) received a second-generation (2G) TKI. RESULTS: Second-generation TKIs were chosen for most patients aged <45 years (69.2%), whereas imatinib was used in 76.7% of patients aged >65 years (p < .001). There was a predominant use of imatinib in intermediate/high European long-term survival risk patients (60.0%/66.0% vs. 49.7% in low-risk patients) and a limited use of 2G-TKIs in patients with comorbidities such as hypertension, diabetes, chronic obstructive pulmonary disease, previous neoplasms, ischemic heart disease, or stroke and in those with >3 concomitant drugs. We observed a greater use of imatinib (61.1%) in patients diagnosed in 2018-2019 compared to 2012-2017 (53.2%; p = .002). In multivariable analysis, factors correlated with imatinib use were age > 65 years, spleen size, the presence of comorbidities, and ≥3 concomitant medications. CONCLUSIONS: This observational study of almost 2000 cases of CML shows that imatinib is the frontline drug of choice in 55% of Italian patients with CP-CML, with 2G-TKIs prevalently used in younger patients and in those with no concomitant clinical conditions. Introduction of the generic formulation in 2018 seems to have fostered imatinib use.

Blinatumomab differentially modulates peripheral blood and bone marrow immune cell repertoire: A Campus ALL study.

Blinatumomab is the first bi-specific T-cell engager approved for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia (B-ALL). Despite remarkable clinical results, the effects of blinatumomab on the host immune cell repertoire are not fully elucidated. In the present study, we characterized the peripheral blood (PB) and, for the first time, the bone marrow (BM) immune cell repertoire upon blinatumomab treatment. Twenty-nine patients with B-ALL received blinatumomab according to clinical practice. Deep multiparametric flow cytometry was used to characterize lymphoid subsets during the first treatment cycle. Blinatumomab induced a transient redistribution of PB effector T-cell subsets and Treg cells with a persistent increase in cytotoxic NK cells, which was associated with a transient upregulation of immune checkpoint receptors on PB CD4 and CD8 T-cell subpopulations and of CD39 expression on suppressive Treg cells. Of note, BM immune T-cell subsets showed a broader post-treatment subversion, including the modulation of markers associated with a T-cell-exhausted phenotype. In conclusion, our study indicates that blinatumomab differentially modulates the PB and BM immune cell repertoire, which may have relevant clinical implications in the therapeutic setting.

Incidence, treatment and outcome of central nervous system relapse in adult acute lymphoblastic leukaemia patients treated front-line with paediatric-inspired regimens: A retrospective multicentre Campus ALL study.

Within the Campus ALL network we analyzed the incidence, characteristics, treatment and outcome of a central nervous system (CNS) relapse in 1035 consecutive adult acute lymphoblastic leukemia (ALL) patients treated frontline with pediatric-inspired protocols between 2009 and 2020. Seventy-one patients (6.8%) experienced a CNS recurrence, more frequently in T- (28/278; 10%) than in B-ALL (43/757; 5.7%) (p = 0.017). An early CNS relapse-< 12 months from diagnosis-was observed in 41 patients. In multivariate analysis, risk factors for early CNS relapse included T-cell phenotype (p = <0.001), hyperleucocytosis >100 × 109 /L (p<0.001) and male gender (p = 0.015). Treatment was heterogeneous, including chemotherapy, radiotherapy, intrathecal therapy and novel agents. A complete remission (CR) was obtained in 39 patients (55%) with no differences among strategies. After CR, 26 patients underwent an allogenic transplant, with a significant overall survival benefit compared to non-transplanted patients (p = 0.012). After a median observation of 8 months from CNS relapse, 23 patients (32%) were alive. In multivariate analysis, the time to CNS relapse was the strongest predictor of a lower 2-year post-relapse survival (p<0.001). In conclusion, in adult ALL the outcome after a CNS relapse remains very poor. Effective CNS prophylaxis remains the best approach and allogenic transplant should be pursued when possible.

Dasatinib-Blinatumomab for Ph-Positive Acute Lymphoblastic Leukemia in Adults.

BACKGROUND: Outcomes in patients with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) have improved with the use of tyrosine kinase inhibitors. Molecular remission is a primary goal of treatment. METHODS: We conducted a phase 2 single-group trial of first-line therapy in adults with newly diagnosed Ph-positive ALL (with no upper age limit). Dasatinib plus glucocorticoids were administered, followed by two cycles of blinatumomab. The primary end point was a sustained molecular response in the bone marrow after this treatment. RESULTS: Of the 63 patients (median age, 54 years; range, 24 to 82) who were enrolled, a complete remission was observed in 98%. At the end of dasatinib induction therapy (day 85), 29% of the patients had a molecular response, and this percentage increased to 60% after two cycles of blinatumomab; the percentage of patients with a molecular response increased further after additional blinatumomab cycles. At a median follow-up of 18 months, overall survival was 95% and disease-free survival was 88%; disease-free survival was lower among patients who had an IKZF1 deletion plus additional genetic aberrations (CDKN2A or CDKN2B, PAX5, or both [i.e., IKZF1 plus]). ABL1 mutations were detected in 6 patients who had increased minimal residual disease during induction therapy, and all these mutations were cleared by blinatumomab. Six relapses occurred. Overall, 21 adverse events of grade 3 or higher were recorded. A total of 24 patients received a stem-cell allograft, and 1 death was related to transplantation (4%). CONCLUSIONS: A chemotherapy-free induction and consolidation first-line treatment with dasatinib and blinatumomab that was based on a targeted and immunotherapeutic strategy was associated with high incidences of molecular response and survival and few toxic effects of grade 3 or higher in adults with Ph-positive ALL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others; GIMEMA LAL2116 D-ALBA EudraCT number, 2016-001083-11; ClinicalTrials.gov number, NCT02744768.).

Multicenter retrospective analysis of clinical outcome of adult patients with mixed-phenotype acute leukemia treated with acute myeloid leukemia-like or acute lymphoblastic leukemia-like chemotherapy and impact of allogeneic stem cell transplantation: a Campus ALL study.

Mixed-phenotype acute leukemia (MPAL) is a rare disease. Treatment is often similar to that of acute lymphoblastic leukemia (ALL), but the outcome in adults and the role of allogeneic stem cell transplantation (AlloSCT) are not well defined. We report on 77 adult patients diagnosed with MPAL over the last 10 years and treated with a curative intent. Median age was 49 years; 7.6% of cases had a BCR::ABL1 rearrangement. Thirty patients (39%) were treated with an acute myeloid leukemia (AML)-like induction and 47 (61%) with an ALL-like scheme. The complete remission (CR) rate was 67.6% and an ALL-like therapy was associated with a better CR rate (P = 0.048). The median OS was 41.9 months; age ≤ 60 years was associated with a better OS (67 vs 26 months, P = 0.014). An AlloSCT was performed in 50 patients (65%). The 5-year OS of transplanted patients was 54%. The OS post-AlloSCT was better in patients who were minimal residual disease (MRD)-negative prior to transplant (75.8% vs 45.2%, P = 0.06). This study shows that MPAL patients respond better to an ALL-like induction therapy; that consolidation therapy should include, whenever possible, an AlloSCT and that MRD negativity should be a primary endpoint of treatment.

Management of polycythemia vera: A survey of treatment patterns in Italy.

OBJECTIVES: Polycythemia vera (PV) is an acquired clonal hematopoietic stem cell disorder characterized by the overproduction of red blood cells. It has long been underlined that there are differences in treatment patterns in routine practice. Therapeutic strategies have also expanded, and in recent years the JAK1/JAK2 inhibitor ruxolitinib has emerged as a second-line therapeutic option in patients who are intolerant to or resistant to hydroxyurea. Determining the impact of changes on practice patterns is of interest, especially for aspects that lack detailed guidance for management. METHODS: To gain insights into treatment patterns by clinicians treating patients with PV in Italy, we carried out a survey of 60 hematologists and transfusion specialists. The questions covered: treatment of low-risk patients, definition of significant leukocytosis, splenomegaly and excessive phlebotomies, resistance/intolerance to hydroxyurea, use of ruxolitinib, cytoreductive therapy, and vaccines. RESULTS: In general, the results of the survey indicate that there is a large heterogeneity in management of patients with PV across these areas. CONCLUSIONS: While helping to provide greater understanding of treatment patterns for patients with PV in Italy, our survey highlights the need for additional clinical studies to obtain more precise guidance for the routine care of patients with PV.

Peripheral blasts are associated with responses to ruxolitinib and outcomes in patients with chronic-phase myelofibrosis.

BACKGROUND: The presence of peripheral blasts (PB) is a negative prognostic factor in patients with primary and secondary myelofibrosis (MF) and PB ≥4% was associated with a particularly unfavorable prognosis. Ruxolitinib (RUX) is the JAK1/2 inhibitor most used for treatment of MF-related splenomegaly and symptoms. Its role has not been assessed in correlation with PB. METHODS: In 794 chronic-phase MF patients treated with RUX, we evaluated the impact of baseline percentage of PB on response (spleen and symptoms responses) and outcome (RUX discontinuation-free, leukemia-free, and overall survival). Three subgroups were compared: PB-0 (no PB, 61.3%), PB-4 (PB 1%-4%, 33.5%), and PB-9 (PB 5%-9%, 5.2%). RESULTS: At 3 and 6 months, spleen responses were less frequently achieved by PB-4 (P = .001) and PB-9 (P = .004) compared to PB-0 patients. RUX discontinuation-free, leukemia-free, and overall survival were also worse for PB-4 and PB-9 patients (P = .001, P = .002, and P < .001, respectively). CONCLUSIONS: Personalized approaches beyond RUX monotherapy may be useful in PB-4 and particularly in PB-9 patients.

Deferasirox in the management of iron overload in patients with myelofibrosis treated with ruxolitinib: The multicentre retrospective RUX-IOL study.

Deferasirox (DFX) is used for the management of iron overload (IOL) in many haematological malignancies including myelofibrosis (MF). The 'RUX-IOL' study retrospectively collected 69 MF patients treated with ruxolitinib (RUX) and DFX for IOL to assess: safety, efficacy in term of iron chelation response (ICR) and erythroid response (ER), and impact on overall survival of the combination therapy. The RUX-DFX therapy was administered for a median time of 12.4 months (interquartile range 3.1-71.2). During treatment, 36 (52.2%) and 34 (49.3%) patients required RUX and DFX dose reductions, while eight (11.6%) and nine (13.1%) patients discontinued due to RUX- or DFX-related adverse events; no unexpected toxicity was reported. ICR and ER were achieved by 33 (47.8%) and 32 patients (46.4%) respectively. Thirteen (18.9%) patients became transfusion-independent. Median time to ICR and ER was 6.2 and 2 months respectively. Patients achieving an ER were more likely to obtain an ICR also (p = 0.04). In multivariable analysis, the absence of leukocytosis at baseline (p = 0.02) and achievement of an ICR at any time (p = 0.02) predicted improved survival. In many MF patients, the RUX-DFX combination provided ICR and ER responses that correlated with improved outcome in the absence of unexpected toxicities. This strategy deserves further clinical investigation.

COVID-19 infection in chronic myeloid leukaemia after one year of the pandemic in Italy. A Campus CML report.

Limited information is available on the impact of the COVID-19 pandemic on the management of chronic myeloid leukaemia (CML). The Campus CML network collected retrospective information on 8 665 CML patients followed at 46 centres throughout Italy during the pandemic between February 2020 and January 2021. Within this cohort, we recorded 217 SARS-CoV-2-positive patients (2·5%). Most patients (57%) were diagnosed as having SARS-CoV-2 infection during the second peak of the pandemic (September 2020 to January 2021). The majority (35%) was aged between 50 and 65 years with a male prevalence (73%). Fifty-six percent of patients presented concomitant comorbidities. The median time from CML diagnosis to SARS-CoV-2 infection was six years (three months to 18 years). Twenty-one patients (9·6%) required hospitalization without the need of respiratory assistance, 18 (8·2%) were hospitalized for respiratory assistance, 8 (3·6%) were admitted to an intensive care unit, while 170 (78%) were only quarantined. Twenty-three percent of patients discontinued tyrosine kinase inhibitor (TKI) therapy during the infection. Twelve patients died due to COVID-19 with a mortality rate of 5·5% in the positive cohort and of 0·13% in the whole cohort. We could also document sequelae caused by the SARS-CoV-2 infection and an impact of the pandemic on the overall management of CML patients.

Arterial thrombosis in Philadelphia-negative myeloproliferative neoplasms predicts second cancer: a case-control study.

Patients with Philadelphia-negative myeloproliferative neoplasm (MPN) are prone to the development of second cancers, but the factors associated with these events have been poorly explored. In an international nested case-control study, we recruited 647 patients with carcinoma, nonmelanoma skin cancer, hematological second cancer, and melanoma diagnosed concurrently or after MPN diagnosis. Up to 3 control patients without a history of cancer and matched with each case for center, sex, age at MPN diagnosis, date of diagnosis, and MPN disease duration were included (n = 1234). Cases were comparable to controls for MPN type, driver mutations and cardiovascular risk factors. The frequency of thrombosis preceding MPN was similar for cases and controls (P = .462). Thrombotic events after MPN and before second cancer were higher in cases than in controls (11.6% vs 8.1%; P = .013), because of a higher proportion of arterial thromboses (6.2% vs 3.7%; P = .015). After adjustment for confounders, the occurrence of arterial thrombosis remained independently associated with the risk of carcinoma (odds ratio, 1.97; 95% confidence interval, 1.14-3.41), suggesting that MPN patients experiencing arterial events after MPN diagnosis deserve careful clinical surveillance for early detection of carcinoma. This study was registered at www.clinicaltrials.gov as NCT03745378.

Prospective assessment of NGS-detectable mutations in CML patients with nonoptimal response: the NEXT-in-CML study.

In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

Ruxolitinib rechallenge in resistant or intolerant patients with myelofibrosis: Frequency, therapeutic effects, and impact on outcome.

BACKGROUND: After ruxolitinib discontinuation, the outcome of patients with myelofibrosis (MF) is poor with scarce therapeutic possibilities. METHODS: The authors performed a subanalysis of an observational, retrospective study (RUX-MF) that included 703 MF patients treated with ruxolitinib to investigate 1) the frequency and reasons for ruxolitinib rechallenge, 2) its therapeutic effects, and 3) its impact on overall survival. RESULTS: A total of 219 patients (31.2%) discontinued ruxolitinib for ≥14 days and survived for ≥30 days. In 60 patients (27.4%), ruxolitinib was rechallenged for ≥14 days (RUX-again patients), whereas 159 patients (72.6%) discontinued it permanently (RUX-stop patients). The baseline characteristics of the 2 cohorts were comparable, but discontinuation due to a lack/loss of spleen response was lower in RUX-again patients (P = .004). In comparison with the disease status at the first ruxolitinib stop, at its restart, there was a significant increase in patients with large splenomegaly (P < .001) and a high Total Symptom Score (TSS; P < .001). During the rechallenge, 44.6% and 48.3% of the patients had spleen and symptom improvements, respectively, with a significant increase in the number of patients with a TSS reduction (P = .01). Although the use of a ruxolitinib dose > 10 mg twice daily predicted better spleen (P = .05) and symptom improvements (P = .02), the reasons for/duration of ruxolitinib discontinuation and the use of other therapies before rechallenge were not associated with rechallenge efficacy. At 1 and 2 years, 33.3% and 48.3% of RUX-again patients, respectively, had permanently discontinued ruxolitinib. The median overall survival was 27.9 months, and it was significantly longer for RUX-again patients (P = .004). CONCLUSIONS: Ruxolitinib rechallenge was mainly used in intolerant patients; there were clinical improvements and a possible survival advantage in many cases, but there was a substantial rate of permanent discontinuation. Ruxolitinib rechallenge should be balanced against newer therapeutic possibilities.

Next-generation sequencing improves BCR-ABL1 mutation detection in Philadelphia chromosome-positive acute lymphoblastic leukaemia.

BCR-ABL1 kinase domain mutation testing in tyrosine kinase inhibitor (TKI)-resistant Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) patients is routinely performed by Sanger sequencing (SS). Recently, next-generation sequencing (NGS)-based approaches have been developed that afford greater sensitivity and straightforward discrimination between compound and polyclonal mutations. We performed a study to compare the results of SS and NGS in a consecutive cohort of 171 Ph+ ALL patients. At diagnosis, 0/44 and 3/44 patients were positive for mutations by SS and NGS respectively. Out of 47 patients with haematologic resistance, 45 had mutations according to both methods, but in 25 patients NGS revealed additional mutations undetectable by SS. Out of 80 patients in complete haematologic response but with BCR-ABL1 ≥0·1%, 28 (35%) and 52 (65%) were positive by SS and NGS respectively. Moreover, in 12 patients positive by SS, NGS detected additional mutations. NGS resolved clonal complexity in 34 patients with multiple mutations at the same or different codons and identified 35 compound mutations. Our study demonstrates that, in Ph+ ALL on TKI therapy, NGS enables more accurate assessment of mutation status both in patients who fail therapy and in patients with minimal residual disease above 0·1%.

Second primary malignancy in myelofibrosis patients treated with ruxolitinib.

Ruxolitinib (RUX), the first JAK1/JAK2 inhibitor approved for myelofibrosis (MF) therapy, has recently been associated with the occurrence of second primary malignancies (SPMs), mainly lymphomas and non-melanoma skin cancers (NMSCs). We analyzed the incidence, risk factors and outcome of SPMs in 700 MF patients treated with RUX in a real-world context. Median follow-up from starting RUX was 2·9 years. Overall, 80 (11·4%) patients developed 87 SPMs after RUX start. NMSCs were the most common SPMs (50·6% of the cases). Multivariate analysis demonstrated that male sex [hazard ratio (HR): 2·37, 95% confidence interval (95%CI): 1·22-4·60, P = 0·01] and thrombocytosis> 400 × 109 /l at RUX start (HR:1·98, 95%CI: 1·10-4·60, P = 0·02) were associated with increased risk for SPMs. Risk factors for NMSC alone were male sex (HR: 3·14, 95%CI: 1·24-7·92, P = 0·02) and duration of hydroxycarbamide and RUX therapy > 5 years (HR: 3·20, 95%CI: 1·17-8·75, P = 0·02 and HR: 2·93, 95%CI: 1·39-6·17, P = 0·005 respectively). In SPMs excluding NMSCs, male sex (HR: 2·41, 95%CI: 1·11-5·25, P = 0·03), platelet > 400 × 109 /l (HR: 3·30, 95%CI: 1·67-6·50, P = 0·001) and previous arterial thromboses (HR: 3·47, 95%CI: 1·48-8·14, P = 0·004) were shown to be associated with higher risk of SPMs. While it is reassuring that no aggressive lymphoma was documented, active skin surveillance is recommended in all patients and particularly after prolonged hydroxycaramide therapy; oncological screening should be triggered by thrombocytosis and arterial thrombosis, particularly in males.