Measurable Residual Disease and Clonal Evolution in Acute Myeloid Leukemia from Diagnosis to Post-Transplant Follow-Up: The Role of Next-Generation Sequencing.

It has now been ascertained that acute myeloid leukemias-as in most type of cancers-are mixtures of various subclones, evolving by acquiring additional somatic mutations over the course of the disease. The complexity of leukemia clone architecture and the phenotypic and/or genotypic drifts that can occur during treatment explain why more than 50% of patients-in hematological remission-could relapse. Moreover, the complexity and heterogeneity of clone architecture represent a hindrance for monitoring measurable residual disease, as not all minimal residual disease monitoring methods are able to detect genetic mutations arising during treatment. Unlike with chemotherapy, which imparts a relatively short duration of selective pressure on acute myeloid leukemia clonal architecture, the immunological effect related to allogeneic hematopoietic stem cell transplant is prolonged over time and must be overcome for relapse to occur. This means that not all molecular abnormalities detected after transplant always imply inevitable relapse. Therefore, transplant represents a critical setting where a measurable residual disease-based strategy, performed during post-transplant follow-up by highly sensitive methods such as next-generation sequencing, could optimize and improve treatment outcome. The purpose of our review is to provide an overview of the role of next-generation sequencing in monitoring both measurable residual disease and clonal evolution in acute myeloid leukemia patients during the entire course of the disease, with special focus on the transplant phase.

BCR::ABL1 levels at first month after TKI discontinuation predict subsequent maintenance of treatment-free remission: A study from the "GRUPPO TRIVENETO LMC".

We analyzed BCR::ABL1 expression at stop and in the first month after discontinuation in 168 chronic myeloid leukemia patients who stopped imatinib or 2nd generation tyrosine kinase inhibitors (2G-TKIs) while in sustained deep molecular response. Patients were divided among those who maintained response (group 1, n = 123) and those who lost major molecular response (group 2, n = 45). Mean BCR::ABL1 RNA levels 1 month after discontinuation were higher in group 2 than in group 1 (p = 0.0005) and the difference was more evident 2 months after stop (p < 0.0001). The same trend was found both for imatinib and 2G-TKIs. A receiver operating characteristic (ROC) analysis to determine a threshold value of BCR::ABL1 at 1 month after discontinuation identified a cut-off value of 0.0051%, with 92.2% specificity, 31.7% sensitivity and a likelihood ratio of 4.087.

Making Treatment-Free Remission (TFR) Easier in Chronic Myeloid Leukemia: Fact-Checking and Practical Management Tools.

In chronic-phase chronic myeloid leukemia (CML), tyrosine kinase inhibitors (TKIs) are the standard of care, and treatment-free remission (TFR) following the achievement of a stable deep molecular response (DMR) has become, alongside survival, a primary goal for virtually all patients. The GIMEMA CML working party recently suggested that the possibility of achieving TFR cannot be denied to any patient, and proposed specific treatment policies according to the patient's age and risk. However, other international recommendations (including 2020 ELN recommendations) are more focused on survival and provide less detailed suggestions on how to choose first and subsequent lines of treatment. Consequently, some grey areas remain. After literature review, a panel of Italian experts discussed the following controversial issues: (1) early prediction of DMR and TFR: female sex, non-high disease risk score, e14a2 transcript and early MR achievement have been associated with stable DMR, but the lack of these criteria is not sufficient to exclude any patient from TFR; (2) criteria for first and subsequent line therapy choice: a number of patient and drug characteristics have been proposed to make a personalized decision; (3) monitoring of residual disease after discontinuation: after the first 6 months, the frequency of molecular tests can be reduced based on MR4.5 persistence and short turnaround time; (4) prognosis of TFR: therapy and DMR duration are important to predict TFR; although immunological control of CML plays a role, no immunological predictive phenotype is currently available. This guidance is intended as a practical tool to support physicians in decision making.

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.

Outcome of very elderly chronic myeloid leukaemia patients treated with imatinib frontline.

Very elderly (> 75 years) chronic myeloid leukaemia (CML) patients at diagnosis are sometimes treated with different doses of imatinib (IM) based on concomitant diseases and physicians' judgement. However, data on long-term follow-up of these patients are still lacking. To investigate treatment response and outcome, we retrospectively revised an Italian database of 263 very elderly CML patients receiving IM from the time of diagnosis. Median age at diagnosis was 78.5 years and 56% of patients had 2 or 3 comorbidities. A complete haematological and cytogenetic response were achieved in 244 (92.8%) and 184 (69.9%) patients, respectively. In 148 cases (56.2%), a major molecular response was observed, which was deep in 63 cases (24%). A blastic phase occurred in 11 patients (4.2%). After a median follow-up of 45.0 months, 93 patients have died (9 from disease progression) and 104 (39.5%) are still in treatment with IM. Incidence of grades 3-4 haematological and non-haematological toxicity was similar to those reported in younger patients. Five-year event-free survival was 54.5% and 45.2% in patients ≤ 80 years and > 80 years, respectively (p = 0.098). Five years OS was 75.7% and 61.6% in patients ≤80 years and > 80 years, respectively (p = 0.003). These findings show that IM plays an important role in frontline treatment of very elderly CML patients without increased toxicity and any effort to treat these patients with standard doses should be made in order to achieve responses as in younger subjects.

Excellent outcomes of 2G-TKI therapy after imatinib failure in chronic phase CML patients.

Second-generation tyrosine kinase inhibitors (2G-TKIs) dasatinib and nilotinib produced historical rates of about 50% complete cytogenetic response (CCyR) and about 40% major molecular response (MMR) in chronic myeloid leukaemia (CML) patients failing imatinib. Direct comparisons between dasatinib and nilotinib are lacking, and few studies addressed the dynamics of deep molecular response (DMR) in a "real-life" setting. We retrospectively analyzed 163 patients receiving dasatinib (n = 95) or nilotinib (n = 68) as second-line therapy after imatinib. The two cohorts were comparable for disease's characteristics, although there was a higher rate of dasatinib use in imatinib-resistant and of nilotinib in intolerant patients. Overall, 75% patients not in CCyR and 60% patients not in MMR at 2G-TKI start attained this response. DMR was achieved by 61 patients (37.4%), with estimated rate of stable DMR at 5 years of 24%. After a median follow-up of 48 months, 60% of patients persisted on their second-line treatment. Rates and kinetics of cytogenetic and molecular responses, progression-free and overall survival were similar for dasatinib and nilotinib. In a "real-life" setting, dasatinib and nilotinib resulted equally effective and safe after imatinib failure, determining high rates of CCyR and MMR, and a significant chance of stable DMR, a prerequisite for treatment discontinuation.

Imatinib and polypharmacy in very old patients with chronic myeloid leukemia: effects on response rate, toxicity and outcome.

BACKGROUND: About 40% of all patients with chronic myeloid leukemia are currently old or very old. They are effectively treated with imatinib, even though underrepresented in clinical studies. Furthermore, as it happens in the general population, they often receive multiple drugs for associated chronic illnesses. Aim of this study was to assess whether or not in imatinib-treated patients aged >75 years the exposure to polypharmacy (5 drugs or more) had an impact on cytogenetic and molecular response rates, event-free and overall survival, as well as on hematological or extra-hematological toxicity. METHODS: 296 patients at 35 Italian hematological institutions were evaluated. RESULTS: Polypharmacy was reported in 107 patients (36.1%), and drugs more frequently used were antiplatelets, diuretics, proton pump inhibitors, ACE-inhibitors, beta-blockers, calcium channel blockers, angiotensin II receptors blockers, statins, oral hypoglycemic drugs and alpha blockers. Complete cytogenetic response was obtained in 174 patients (58.8%), 78 (26.4%) within 6 month, 63 (21.3%) between 7 and 12 months. Major molecular response was obtained in 153 patients (51.7%), 64 (21.6%) within the 12 month. One hundred and twenty-eight cases (43.2%) of hematological toxicity were recorded, together with 167 cases (56.4%) of extra-hematological toxicity. Comparing patients exposed to polypharmacy to those without, no difference was observed pertaining to the dosage of imatinib, cytogenetic and molecular responses and hematological and extra-hematological toxicity. CONCLUSION: Notwithstanding the several interactions reported in the literature between imatinib and some of the medications considered herewith, this fact does not seem to have a clinical impact on response rate and outcome.

EUTOS score predicts long-term outcome but not optimal response to imatinib in patients with chronic myeloid leukaemia.

To test the recently developed EUTOS score in predicting optimal response to imatinib and the long-term outcome, 265 patients with early chronic phase chronic myeloid leukaemia treated with standard dose imatinib were analysed. Achievement of optimal response endpoints were higher in low-risk patients, though the difference was not statistically significant: PCyR at 6th month 86% vs 67% (p=0.06), CCyR at 12th month 80% vs 63% (p=0.09), MMR at 18th month 61% vs 36% (p=0.11). However, EUTOS score was predictive for the long-term response. With a median follow-up of 61 months, 53% high-risk patients experienced imatinib failure, compared to 23% in the low-risk group (p=0.013). Among high-risk patients, 4/17 (23%) progressed to accelerated/blastic phase or died, compared to 11/248 (5%) low-risk patients, with 5-year progression-free survival rates of 84±10% and 96±1%, respectively (p=0.04). Our data confirm that EUTOS score envisions the long-term outcome of imatinib therapy.

Imatinib in very elderly patients with chronic myeloid leukemia in chronic phase: a retrospective study.

BACKGROUND: A large number of chronic myeloid leukemia (CML) patients are treated with imatinib mesylate outside of clinical trials, which may not be representative of common clinical practice. The age of CML patients enrolled within controlled clinical studies is lower with respect to patients included in population-based registries. PATIENTS AND METHODS: To describe the safety and tolerability of imatinib in very elderly CML patients in chronic phase, 211 chronic-phase CML patients aged >75 years were retrospectively analyzed using data collected from 31 institutions in Italy. RESULTS: The median age at imatinib start was 78.6 years [interquartile range (IR) 76.3-81.4], median time from diagnosis to imatinib start was 1.2 months (IR 0.5-3.7). The starting dose of imatinib was 400 mg/day in 144 patients (68.2 %), >400 mg/day in 4 patients (2.0 %), and <400 mg/day in 63 patients (29.8 %); overall, 94 patients (44.5 %) needed a dose reduction and 27 (12.7 %) discontinued imatinib for toxicity. Grade 3-4 hematologic and extrahematologic toxicities were observed in 40 (18.9 %) and 45 (21.3 %) patients, respectively. After a median observation of 29.8 months (IR 13.0-55.6), 203/211 patients had at least 6 months of observation on imatinib or discontinued before and were evaluable for response and outcome; of them, 183 patients (90.2 %) achieved a complete hematologic response (CHR). Among these 183 patients in CHR, 14 refused any other karyotypic or molecular evaluation, 24 achieved CHR only, and 145 (71.4 %) achieved a cytogenetic response (CyR) of any grade, which was complete (CCyR) in 129 (63.5 %). Among the 129 patients with CCyR, 95 (46.7 %) achieved a major molecular response (MMolR). By multivariate regression analysis, late chronic phase (p = 0.001) and grade 3-4 extrahematologic toxicity (p = 0.007) maintained a negative independent prognostic impact for CCyR, while late chronic phase (p = 0.026), grade 3-4 extrahematologic toxicity (p = 0.007), and lower initial dose of imatinib (p = 0.044) maintained a negative independent prognostic impact for MMolR. The 2-year and 4-year overall survival were 92.6 % (95 % CI 88.7-96.5) and 78.0 % (95 % CI 71.2-84.8), respectively. CONCLUSIONS: Results from this large cohort of patients show that no upper age limit should be applied for the administration of imatinib to patients with chronic-phase CML; the very elderly, including those with concomitant severe diseases, should be offered this treatment. The role of a reduced starting dose of imatinib warrants further studies.

Incidence, risk factors and management of pleural effusions during dasatinib treatment in unselected elderly patients with chronic myelogenous leukaemia.

To assess the most important features and clinical impact of pleural effusions, which are a common toxicity during dasatinib treatment and often impair its high efficacy, 172 unselected consecutive patients with chronic myelogenous leukaemia in chronic phase treated in 27 Italian centres, with dasatinib when aged >60 years for resistance/intolerance to imatinib, were examined. During treatment, 52/172 patients (30.2%) presented pleural effusion, which was grades 1-2 in 38 patients and grades 3-4 in 14 patients (8.1% of the entire cohort of patients), according to the WHO scale; in 14/52 patients (26.9%), there was a concomitant pericardial effusion. Pleural effusion was recurrent in 25/52 patients (48.0%). Median time from dasatinib to first pleural effusion was 11.0 months (interquartile range 3.6-18.6). Eleven patients (6.4%) required permanent dasatinib discontinuation. Only presence of concomitant pulmonary disease ( p = 0.035) and initial daily dose of dasatinib (140 mg vs 100 mg, p = 0.014) were significantly associated with pleural effusions. There were no differences among patients with or without pleural effusions as concerns response rates and overall survival. Pleural effusions were common in our unselected 'real-life' population of elderly patients but were clinically manageable and did not seem to affect treatment results.

The role of MDR-related proteins in the prognosis of adult acute myeloid leukaemia (AML) with normal karyotype.

Cytogenetic abnormalities are among the most important factors affecting the outcome of patients with acute myeloid leukaemia (AML), but approximately 40-50% of AML cases display a normal karyotype at diagnosis. Multidrug resistance (MDR) proteins overexpression is associated with worse prognosis in acute leukaemias, but its role in normal karyotype AML is less defined. We analysed the expression of P-glycoprotein (PGP), MDR-related protein (MRP) and lung resistance protein (LRP) in 135 adult patients with normal karyotype AML and its correlation with other biological features of the disease, to evaluate the impact of MDR proteins on response to therapy and on survival. Increased PGP expression was associated with lower rate of complete remission (CR; p = 0.006), similarly to advanced age. Cases overexpressing PGP displayed also a shorter event-free survival (EFS; 4 vs. 10 months, p = 0.035) and the increased expression of at least one MDR protein was associated with a reduced overall survival (OS; p = 0.038). Also age was predictive of worse prognosis. Our data confirm the prognostic role of MDR proteins, in particular of PGP, also in AML patients with normal karyotype at diagnosis. This finding could be used to stratify patients with different prognosis and to design risk-adapted therapeutic strategies.

The prognostic value of P-glycoprotein (ABCB) and breast cancer resistance protein (ABCG2) in adults with de novo acute myeloid leukemia with normal karyotype.

Multidrug resistance is a major cause of treatment failure in acute myeloid leukemia (AML). P-glycoprotein (PGP) over-expression has an unfavorable prognostic significance, while the role of breast cancer resistance protein (BCRP) is less clear, especially in AML patients with a normal karyotype. We studied 73 consecutive AML patients with a normal karyotype. BCRP was over-expressed in 24 patients (33%) and was significantly co-expressed with PGP (13/24 vs 11/49, p=0.006) and with CD56. Only PGP, along with age and CD34, affected the achievement of complete remission (p=0.02), while BCRP-positive cases showed an increased risk of relapse (p=0.005) and a shorter disease-free survival (p=0.027). BCRP over-expression did not influence the achievement of remission, but significantly affected the duration of complete remissions. BCRP may, therefore, be regarded as a prognostic factor in patients with normal karyotype AML, for the design of risk-adapted post-remission therapy.

Epstein-Barr virus reactivation in a patient treated with anti-thymocyte globulin for severe aplastic anemia.

Epstein-Barr virus (EBV) infection and reactivation is an increasing complication in immune deficient patients, particularly after allogeneic hematopoietic stem cell transplantation (HSCT). Therapy with anti-thymocyte globulin (ATG) is associated with higher incidence of EBV-related disease in HSCT patients, but this risk is not documented in patients receiving ATG for severe aplastic anemia (SAA). We describe the case of a patient who developed an EBV infection, with the clinical features of an infectious mononucleosis, after immune suppression with cyclosporine and two courses of ATG for SAA.

The development of autoantibodies after allogeneic stem cell transplantation is related with chronic graft-vs-host disease and immune recovery.

OBJECTIVE: Chronic graft-vs-host disease (GVHD) has certain similarities with autoimmune diseases and is associated with the development of various autoantibodies in some patients. In this study, we analyzed the occurrence of autoantibodies in 63 patients surviving longer than 3 months after an allogeneic haematopoietic stem cell transplantation (HSCT), with the aim of detecting a possible association between occurrence of autoantibodies and development of chronic GVHD and immune recovery after HSCT. PATIENTS AND METHODS: The patients were screened every 3 months for the occurrence of the following autoantibodies: anti-nuclear (ANA), anti-mitochondrial (AMA), anti-smooth muscle (ASMA), anti-cardiolipin (ACLA), anti-liver-kidney microsomal (LKM), anti-DNA, anti-neutrophil cytoplasmatic (ANCA), and anti-thyroid antibodies. Peripheral blood immunophenotyping with anti-CD3, CD4, CD8, CD19, CD20, CD16, and CD56 antibodies was evaluated at the same intervals. RESULTS: Autoantibodies were not found in 18 patients (29%), at least in one screening in 29 patients (46%), and in all screenings in 16 patients (25%). ANA were found in 41 patients (65%), AMA in 4 (6%), ASMA in 4 (6%), ANCA in 7 (11%), ACLA in 1 (2%), anti-thyroid antibodies in 3 (5%), and anti-DNA in 2 (3%). More than one antibody occurred in 16/63 (25%) positive patients. ANA was significantly more frequent in patients with chronic GVHD and, among these, in those with the extensive form. The nucleolar pattern of immunofluorescence of ANA but not its titer was correlated with the extension of chronic GVHD. Patients who developed autoantibodies had higher CD20(+) cell blood counts than negative patients in the third month (p=0.006), ninth month (p=0.061), and twelfth month (p=0.043). CONCLUSION: We conclude that patients with chronic GVHD, particularly those with an extensive involvement, were likely to develop autoantibodies and have a faster B-cell recovery, suggesting a role of B cells in the pathogenesis of chronic GVHD.

Effect of amifostine on the cytotoxicity of daunorubicin and daunoxome in tumor and normal cells.

Anthracyclines are powerful cytotoxic agents, used as first-line treatment of leukemias and many other tumors, but host-tissue toxicity is their main dose-limiting factor. However, their therapeutic effects depend not only on the toxicity, hence on the dose, but also on drug resistance. Among the mechanisms that can account for cell sensitivity to anthracyclines, there is an overexpression of drug transport proteins, like the transmembrane P-glycoprotein (PGP), the multidrug- resistance-related protein (MRP) and the lung-resistance-related protein (LRP). Attempts to reduce the toxicity of chemotherapeutic agents without affecting their efficacy have been made using liposomal anthracyclines or cytoprotective agents, as Amifostine. The aim of this study was to evaluate and compare the toxic effects of Daunorubicin, in normal or liposomal formulation, used in combination with WR1065, the active metabolite of Amifostine, against normal and tumor cells. In conclusion these data show that the preincubation with WR-1065 does not inhibit the drug toxic effect on blast cells and on tumor cell lines, independently by their multidrug resistance phenotype, but has a cytoprotective effect on stem cells causing a drug cytotoxicity reduction of 10-20%. This advantage is even higher using the liposomal formulation of DNR. Therefore, Amifostine can offer a chance of protecting normal cells from the toxicity of anthracyclines, in normal or liposomal formulation. The combination of liposomal anthracyclines with Amifostine can confer further advantages in management of leukemic patients, especially the elderly where treatment toxicity is a main problem. These patients may be candidates for alternative therapeutic strategies and the combination of DNX and Amifostine is an attractive treatment for these cases where a low nonhematological toxicity is required.