Publications by authors named "Giuseppe Saglio"

314 Publications

Shedding Light on Targeting Chronic Myeloid Leukemia Stem Cells.

J Clin Med 2021 Dec 11;10(24). Epub 2021 Dec 11.

Department of Clinical Biological Sciences, University of Turin, San Luigi University Hospital, 10043 Turin, Italy.

Chronic myeloid leukemia stem cells (CML LSCs) are a rare and quiescent population that are resistant to tyrosine kinase inhibitors (TKI). When TKI therapy is discontinued in CML patients in deep, sustained and apparently stable molecular remission, these cells in approximately half of the cases restart to grow, resuming the leukemic process. The elimination of these TKI resistant leukemic stem cells is therefore an essential step in increasing the percentage of those patients who can reach a successful long-term treatment free remission (TFR). The understanding of the biology of the LSCs and the identification of the differences, phenotypic and/or metabolic, that could eventually allow them to be distinguished from the normal hematopoietic stem cells (HSCs) are therefore important steps in designing strategies to target LSCs in a rather selective way, sparing the normal counterparts.
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http://dx.doi.org/10.3390/jcm10245805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8708315PMC
December 2021

Droplet Digital PCR for Monitoring in Diagnostic Routine: Ready to Start?

Cancers (Basel) 2021 Oct 30;13(21). Epub 2021 Oct 30.

Department of Clinical and Biological Sciences, University of Turin, AOU San Luigi Gonzaga, Orbassano, 10043 Turin, Italy.

mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDx %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of % IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.
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http://dx.doi.org/10.3390/cancers13215470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8582412PMC
October 2021

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

Br J Haematol 2021 Oct 11. Epub 2021 Oct 11.

Ematologia, Azienda Ospedaliera Universitaria - Università degli Studi di Napoli 'Federico II', Napoli, Italy.

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.
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http://dx.doi.org/10.1111/bjh.17890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8652631PMC
October 2021

Prognostic Factors for Overall Survival In Chronic Myeloid Leukemia Patients: A Multicentric Cohort Study by the Italian CML GIMEMA Network.

Front Oncol 2021 26;11:739171. Epub 2021 Aug 26.

Haematology Unit, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza, Torino, Italy.

An observational prospective study was conducted by the CML Italian network to analyze the role of baseline patient characteristics and first line treatments on overall survival and CML-related mortality in 1206 newly diagnosed CML patients, 608 treated with imatinib (IMA) and 598 with 2 generation tyrosine kinase inhibitors (2GTKI). IMA-treated patients were much older (median age 69 years, IQR 58-77) than the 2GTKI group (52, IQR 41-63) and had more comorbidities. Estimated 4-year overall survival of the entire cohort was 89% (95%CI 85.9-91.4). Overall, 73 patients (6.1%) died: 17 (2.8%) in the 2GTKI vs 56 (9.2%) in the IMA cohort (adjusted HR=0.50; 95% CI=0.26-0.94), but no differences were detected for CML-related mortality (10 (1.7%) vs 11 (1.8%) in the 2GTKIs vs IMA cohort (sHR=1.61; 0.52-4.96). The ELTS score was associated to CML mortality (high risk vs low, HR=9.67; 95%CI 2.94-31.74; p<0.001), while age (per year, HR=1.03; 95%CI 1.00-1.06; p=0.064), CCI (4-5 vs 2, HR=5.22; 95%CI 2.56-10.65; p<0.001), ELTS score (high risk vs low, HR=3.11; 95%CI 1.52-6.35, p=0.002) and 2GTKI vs IMA (HR=0.26; 95%CI 0.10-0.65, p=0.004) were associated to an increased risk of non-related CML mortality. The ELTS score showed a better discriminant ability than the Sokal score in all comparisons.
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http://dx.doi.org/10.3389/fonc.2021.739171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427308PMC
August 2021

JAK Inhibition with Ruxolitinib in Patients with COVID-19 and Severe Pneumonia: Multicenter Clinical Experience from a Compassionate Use Program in Italy.

J Clin Med 2021 Aug 23;10(16). Epub 2021 Aug 23.

Department of Clinical and Biological Sciences of the University of Turin, San Luigi University Hospital, 10143 Orbassano, Italy.

Jak inhibitors are potent anti-inflammatory drugs that have the potential to dampen the hyperactive inflammatory response associated with severe COVID-19. We reviewed the clinical outcomes of 218 patients with COVID-19 hospitalized for severe pneumonia and treated with ruxolitinib through a compassionate use program. Data on the duration of treatment; outcomes at 4, 7, 14, and 28 days; oxygen support requirements; clinical status; and laboratory parameters were retrospectively collected. Overall, according to the physician evaluation, 66.5% of patients showed improvement at follow-up; of these, 83.5% showed improvement by day 7. Oxygen support status also showed improvement, and by day 7, 21.6% of patients were on ambient air, compared with 1.4% at baseline, which increased to 48.2% by day 28. Significant decreases in C-reactive protein and increases in the lymphocyte total count were already observed by day 4, which seemed to correlate with a positive outcome. At the end of the observation period, 87.2% of patients were alive. No unexpected safety findings were observed, and grade 3/4 adverse events were reported in 6.9% of patients.
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http://dx.doi.org/10.3390/jcm10163752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397012PMC
August 2021

Alignment of Qx100/Qx200 Droplet Digital (Bio-Rad) and QuantStudio 3D (Thermofisher) Digital PCR for Quantification of BCR-ABL1 in Ph+ Chronic Myeloid Leukemia.

Diseases 2021 May 5;9(2). Epub 2021 May 5.

Unit of Blood Diseases and Stem Cell Transplantation, DPT of Clinical and Experimental Sciences, University of Brescia, ASST SpedaliCivili di Brescia, 25123 Brescia, Italy.

In recent years, the digital polymerase chain reaction has received increasing interest as it has emerged as a tool to provide more sensitive and accurate detection of minimal residual disease. In order to start the process of data alignment, we assessed the consistency of the BCR-ABL1 quantification results of the analysis of 16 RNA samples at different levels of disease. The results were obtained by two different laboratories that relied on The Qx100/Qx200 Droplet Digital PCR System (Bio-Rad) and Quant Studio 3D dPCR System (Thermofisher) platforms. We assessed the compatibility between the estimated values by linear regression, Bland-Altman bias-plot, and Mann-Whitney nonparametric test. The results confirmed the compatibility of the measures, allowing us tocompute an 'alignment factor' (AF), equal to 1.41, which was further validated by a different series of experiments. We conclude that the performed measurements by the two laboratories are comparable, and also equalized through the introduction of an alignment factor.
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http://dx.doi.org/10.3390/diseases9020035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161814PMC
May 2021

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-]pyridine Scaffold: SAR of the Biphenyl Moiety.

J Med Chem 2021 05 12;64(9):5404-5428. Epub 2021 Apr 12.

Department of Drug Science and Technology, University of Turin, Via P. Giuria 9, Turin 10125, Italy.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (DHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound , a potent DHODH inhibitor (IC = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC = 265 nM). Herein, we investigate the drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, is characterized by higher potency in inducing myeloid differentiation (EC = 17.3 nM), strong proapoptotic properties (EC = 20.2 nM), and low cytotoxicity toward non-AML cells (EC(Jurkat) > 100 μM).
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http://dx.doi.org/10.1021/acs.jmedchem.0c01549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279415PMC
May 2021

Targeting Chronic Myeloid Leukemia Stem/Progenitor Cells Using Venetoclax-Loaded Immunoliposome.

Cancers (Basel) 2021 Mar 15;13(6). Epub 2021 Mar 15.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

CML is a hematopoietic stem-cell disorder emanating from breakpoint cluster region/Abelson murine leukemia 1 (BCR/ABL) translocation. Introduction of different TKIs revolutionized treatment outcome in CML patients, but CML LSCs seem insensitive to TKIs and are detectable in newly diagnosed and resistant CML patients and in patients who discontinued therapy. It has been reported that CML LSCs aberrantly express some CD markers such as CD26 that can be used for the diagnosis and for targeting. In this study, we confirmed the presence of CD26+ CML LSCs in newly diagnosed and resistant CML patients. To selectively target CML LSCs/progenitor cells that express CD26 and to spare normal HSCs/progenitor cells, we designed a venetoclax-loaded immunoliposome (IL-VX). Our results showed that by using this system we could selectively target CD26+ cells while sparing CD26- cells. The efficiency of venetoclax in targeting CML LSCs has been reported and our system demonstrated a higher potency in cell death induction in comparison to free venetoclax. Meanwhile, treatment of patient samples with IL-VX significantly reduced CD26+ cells in both stem cells and progenitor cells population. In conclusion, this approach showed that selective elimination of CD26+ CML LSCs/progenitor cells can be obtained in vitro, which might allow in vivo reduction of side effects and attainment of treatment-free, long-lasting remission in CML patients.
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http://dx.doi.org/10.3390/cancers13061311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000981PMC
March 2021

Dosing Strategies for Improving the Risk-Benefit Profile of Ponatinib in Patients With Chronic Myeloid Leukemia in Chronic Phase.

Front Oncol 2021 16;11:642005. Epub 2021 Mar 16.

Hematology, Department of Translational and Precision Medicine, Sapienza University, Rome, Italy.

The treatment of chronic myeloid leukemia (CML) has been advanced by the development of small-molecule tyrosine kinase inhibitors (TKIs), which target the fusion protein BCR-ABL1 expressed by the Philadelphia chromosome. Ponatinib is a 3rd generation TKI that binds BCR-ABL1 with high affinity and inhibits most BCR-ABL1 mutants, including the T315I mutation. The approved starting dose of ponatinib is 45 mg once daily (full dose), however, the need for a full dose, especially in patients with dose adjustments due to tolerability problems, remains undemonstrated. Lower starting doses of ponatinib (30 mg or 15 mg once daily) for patients "with lesser degrees of resistance or multiple intolerances, especially those with an increased cardiovascular risk profile" has been recommended by the 2020 European LeukemiaNet. However, the available literature and guidance on the use of ponatinib at low dosage are limited. The objective of this paper is to describe how we select ponatinib dosage for CML patients in chronic phase in our clinical practice based on the available evidence and our clinical experience. We propose dosing regimens for the optimal starting dose for six generic cases of CML patients in chronic phase eligible for the switch to ponatinib and provide an algorithm to guide ponatinib dosing during treatment.
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http://dx.doi.org/10.3389/fonc.2021.642005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009177PMC
March 2021

Prognostic Impact of Baseline Immunologic Profile in Aggressive B-cell non-Hodgkin's Lymphomas.

Mediterr J Hematol Infect Dis 2021 1;13(1):e2021018. Epub 2021 Mar 1.

Division of Onco-Hematoncology, European Institute of Oncology, IRCCS, Milano, Italy.

Host immune homeostasis as an independent prognostic indicator has been inadequately evaluated in aggressive non-Hodgkin's lymphomas (NHL). The present study addresses the prognostic significance in aggressive NHLs of the immunologic profile evaluated by pretreatment serum levels of immunoglobulins (Ig) and lymphocyte-monocyte ratio (LMR). In this series of 90 patients with aggressive lymphoma, the median level for IgG was 1,024mg/dl (range 436-2236), and for LMR was 2.2 (range 0.2-13.8). CR rate was higher with IgG levels ≥1,024mg/dL (91% vs 77% p=0.059). LMR ≤ 2.2 was associated with lower 1-year PFS (73% vs. 92%, p 0.016). Patients with good/very good R-IPI showed a reduced PFS if IgG or LMR was low, while patients with poor R-IPI did better if LMR or IgG levels were high. We combined both parameters with the R-IPI and produced a four-risk prognostic score showing one-year PFS of 95% (95% CI 68%-99%), 100% (95% CI 100%-100%), 73% (95% CI 52%-86%), and 59% (95% CI 31%-79%), in patients with zero, one, two and three risk factors, respectively. The results indicate for the first time the value of baseline serum Ig levels in the prognostic assessment of aggressive lymphoma.
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http://dx.doi.org/10.4084/MJHID.2021.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938923PMC
March 2021

Treatment-free remission following frontline nilotinib in patients with chronic phase chronic myeloid leukemia: 5-year update of the ENESTfreedom trial.

Leukemia 2021 05 11;35(5):1344-1355. Epub 2021 Mar 11.

University of Turin, Orbassano, Italy.

The ENESTfreedom trial assessed the feasibility of treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase (CML-CP) following frontline nilotinib treatment. Results for long-term outcomes after a 5-year follow-up are presented herein. Patients who had received ≥2 years of frontline nilotinib therapy and achieved MR underwent a 1-year nilotinib treatment consolidation phase before attempting TFR. At the 5-year data cut-off, 81/190 patients entering the TFR phase (42.6%) were still in TFR, with 76 (40.0%) in MR. Patients who lost major molecular response (MMR) entered a treatment re-initiation phase; 90/91 patients entering this phase (98.9%) regained MMR and 84/91 patients (92.3%) regained MR. The Kaplan-Meier estimated treatment-free survival rate at 5 years was 48.2%. No disease progression or CML-related deaths were reported. Whereas the incidence of adverse events (AEs) declined from 96 weeks following the start of TFR, an increase in AE frequency was observed for patients in the treatment re-initiation phase. Low Sokal risk score, BCR-ABL1 levels at 48 weeks of TFR and stable MR response for the first year of TFR were associated with higher TFR rates. Overall, these results support the efficacy and safety of attempting TFR following upfront nilotinib therapy of >3 years in patients with CML-CP.
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http://dx.doi.org/10.1038/s41375-021-01205-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102196PMC
May 2021

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Cancers (Basel) 2021 Feb 28;13(5). Epub 2021 Feb 28.

Department of Clinical and Biological Sciences, University of Turin, Orbassano, 10043 Turin, Italy.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting activity.
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http://dx.doi.org/10.3390/cancers13051003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957697PMC
February 2021

The Giant HECT E3 Ubiquitin Ligase HERC1 Is Aberrantly Expressed in Myeloid Related Disorders and It Is a Novel BCR-ABL1 Binding Partner.

Cancers (Basel) 2021 Jan 19;13(2). Epub 2021 Jan 19.

Department of Oncology, Medical School, University of Torino, 10043 Orbassano, Italy.

HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of in myeloid related blood disorders.
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http://dx.doi.org/10.3390/cancers13020341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832311PMC
January 2021

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Cancers (Basel) 2021 Jan 14;13(2). Epub 2021 Jan 14.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the () sequence and the () gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the transgenic as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.
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http://dx.doi.org/10.3390/cancers13020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830713PMC
January 2021

Long-term outcomes with frontline nilotinib versus imatinib in newly diagnosed chronic myeloid leukemia in chronic phase: ENESTnd 10-year analysis.

Leukemia 2021 02 7;35(2):440-453. Epub 2021 Jan 7.

Universitätsklinikum Jena, Jena, Germany.

In the ENESTnd study, with ≥10 years follow-up in patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase, nilotinib demonstrated higher cumulative molecular response rates, lower rates of disease progression and CML-related death, and increased eligibility for treatment-free remission (TFR). Cumulative 10-year rates of MMR and MR were higher with nilotinib (300 mg twice daily [BID], 77.7% and 61.0%, respectively; 400 mg BID, 79.7% and 61.2%, respectively) than with imatinib (400 mg once daily [QD], 62.5% and 39.2%, respectively). Cumulative rates of TFR eligibility at 10 years were higher with nilotinib (300 mg BID, 48.6%; 400 mg BID, 47.3%) vs imatinib (29.7%). Estimated 10-year overall survival rates in nilotinib and imatinib arms were 87.6%, 90.3%, and 88.3%, respectively. Overall frequency of adverse events was similar with nilotinib and imatinib. By 10 years, higher cumulative rates of cardiovascular events were reported with nilotinib (300 mg BID, 16.5%; 400 mg BID, 23.5%) vs imatinib (3.6%), including in Framingham low-risk patients. Overall efficacy and safety results support the use of nilotinib 300 mg BID as frontline therapy for optimal long-term outcomes, especially in patients aiming for TFR. The benefit-risk profile in context of individual treatment goals should be carefully assessed.
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http://dx.doi.org/10.1038/s41375-020-01111-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862065PMC
February 2021

A Retrospective Analysis about Frequency of Monitoring in Italian Chronic Myeloid Leukemia Patients after Discontinuation.

J Clin Med 2020 Nov 17;9(11). Epub 2020 Nov 17.

Hematology Unit, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy.

Successful discontinuation of tyrosine kinase inhibitors has been achieved in patients with chronic-phase chronic myeloid leukemia (CML). Careful molecular monitoring after discontinuation warrants safe and prompt resumption of therapy. We retrospectively evaluated how molecular monitoring has been conducted in Italy in a cohort of patients who discontinued tyrosine kinase inhibitor (TKI) treatment per clinical practice. The outcome of these patients has recently been reported-281 chronic-phase CML patients were included in this subanalysis. Median follow-up since discontinuation was 2 years. Overall, 2203 analyses were performed, 17.9% in the first three months and 38.4% in the first six months. Eighty-six patients lost major molecular response (MMR) in a mean time of 5.7 months-65 pts (75.6%) during the first six months. We evaluated the number of patients who would experience a delay in diagnosis of MMR loss if a three-month monitoring schedule was adopted. In the first 6 months, 19 pts (29.2%) would have a one-month delay, 26 (40%) a 2-month delay. Very few patients would experience a delay in the following months. A less intense frequency of monitoring, particularly after the first 6 months off treatment, would not have affected the success of treatment-free remission (TFR) nor put patients at risk of progression.
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http://dx.doi.org/10.3390/jcm9113692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698481PMC
November 2020

Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR.

Cancers (Basel) 2020 Nov 6;12(11). Epub 2020 Nov 6.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized. This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.
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http://dx.doi.org/10.3390/cancers12113287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694607PMC
November 2020

Point: Is there a best duration of deep molecular response to achieve therapy-free remission in chronic myeloid leukaemia?

Br J Haematol 2021 01 10;192(1):22-23. Epub 2020 Nov 10.

Department of Clinical and Biological Sciences, University Hospital San Luigi, University of Turin, Turin, Italy.

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http://dx.doi.org/10.1111/bjh.17112DOI Listing
January 2021

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Int J Mol Sci 2020 Oct 16;21(20). Epub 2020 Oct 16.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

I is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.
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http://dx.doi.org/10.3390/ijms21207674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589297PMC
October 2020

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

J Cell Mol Med 2020 09 13;24(18):10978-10986. Epub 2020 Aug 13.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.
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http://dx.doi.org/10.1111/jcmm.15730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521327PMC
September 2020

A Clinically Applicable Approach to the Classification of B-Cell Non-Hodgkin Lymphomas with Flow Cytometry and Machine Learning.

Cancers (Basel) 2020 Jun 24;12(6). Epub 2020 Jun 24.

Laboratory of Immunopathology, Division of Pathology, A.O. Ordine Mauriziano, 10128 Turin, Italy.

The immunophenotype is a key element to classify B-cell Non-Hodgkin Lymphomas (B-NHL); while it is routinely obtained through immunohistochemistry, the use of flow cytometry (FC) could bear several advantages. However, few FC laboratories can rely on a long-standing practical experience, and the literature in support is still limited; as a result, the use of FC is generally restricted to the analysis of lymphomas with bone marrow or peripheral blood involvement. In this work, we applied machine learning to our database of 1465 B-NHL samples from different sources, building four artificial predictive systems which could classify B-NHL in up to nine of the most common clinico-pathological entities. Our best model shows an overall accuracy of 92.68%, a mean sensitivity of 88.54% and a mean specificity of 98.77%. Beyond the clinical applicability, our models demonstrate (i) the strong discriminatory power of MIB1 and Bcl2, whose integration in the predictive model significantly increased the performance of the algorithm; (ii) the potential usefulness of some non-canonical markers in categorizing B-NHL; and (iii) that FC markers should not be described as strictly positive or negative according to fixed thresholds, but they rather correlate with different B-NHL depending on their level of expression.
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http://dx.doi.org/10.3390/cancers12061684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352227PMC
June 2020

Illuminating novel biological aspects and potential new therapeutic approaches for chronic myeloproliferative malignancies.

Hematol Oncol 2020 Dec 4;38(5):654-664. Epub 2020 Sep 4.

University of California Irvine, Irvine, California, USA.

This review reflects the presentations and discussion at the 14th post-American Society of Hematology (ASH) International Workshop on Chronic Myeloproliferative Malignancies, which took place on the December 10 and 11, 2019, immediately after the 61st ASH Annual Meeting in Orlando, Florida. Rather than present a resume of the proceedings, we address some of the topical translational science research and clinically relevant topics in detail. We consider how recent studies using single-cell genomics and other molecular methods reveal novel aspects of hematopoiesis which in turn raise the possibility of new therapeutic approaches for patients with myeloproliferative neoplasms (MPNs). We discuss how alternative therapies could benefit patients with chronic myeloid leukemia who develop BCR-ABL1 mutant subclones following ABL1-tyrosine kinase inhibitor therapy. In MPNs, we focus on efforts beyond JAK-STAT and the merits of integrating activin receptor ligand traps, interferon-α, and allografting in the current treatment algorithm for patients with myelofibrosis.
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http://dx.doi.org/10.1002/hon.2771DOI Listing
December 2020

First-line imatinib vs second- and third-generation TKIs for chronic-phase CML: a systematic review and meta-analysis.

Blood Adv 2020 06;4(12):2723-2735

Analytical Epidemiology and Health Impact Unit, Department of Research, National Tumour Institute of Milan, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Foundation, Milan, Italy.

Imatinib, the first tyrosine kinase inhibitor (TKI) for the treatment of chronic myeloid leukemia (CML), improves overall survival (OS), but the introduction of newer TKIs requires the definition of the optimal first-line TKI for newly diagnosed Philadelphia chromosome-positive (Ph+) chronic-phase (CP) CML. This systematic review of randomized controlled trials (RCTs) compares the efficacy and safety of imatinib vs second-generation (dasatinib, nilotinib, bosutinib) and third-generation TKIs (ponatinib) in adults with newly diagnosed Ph+ CP CML, concentrating on OS, progression-free survival (PFS), and hematological and nonhematological adverse events. The quality of the evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) method. Seven RCTs published between 1990 and 2019 (involving 3262 participants) satisfied the eligibility criteria. Two RCTs (imatinib vs nilotinib and imatinib vs dasatinib) found no difference in 5-year OS or PFS. Second- and third-generation TKIs improved 3-month major molecular responses (relative risk [RR], 4.28; 95% confidence interval [CI], 2.20-8.32) and other efficacy outcomes, decreased accelerated/blastic-phase transformations (RR, 0.44; 95% CI, 0.26-0.74), but were associated with more cases of thrombocytopenia (RR, 1.57; 95% CI, 1.20-2.05), cardiovascular events (RR, 2.54; 95% CI, 1.49-4.33), and pancreatic (RR, 2.29; 95% CI, 1.32-3.96) and hepatic effects (RR, 3.51; 95% CI 1.55-7.92). GRADE showed that the certainty of the evidence ranged from high to moderate. This study shows that, in comparison with imatinib, second- and third-generation TKIs improve clinical responses, but the safer toxicity profile of imatinib may make it a better option for patients with comorbidities.
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http://dx.doi.org/10.1182/bloodadvances.2019001329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322957PMC
June 2020

Iron overload alters the energy metabolism in patients with myelodysplastic syndromes: results from the multicenter FISM BIOFER study.

Sci Rep 2020 06 8;10(1):9156. Epub 2020 Jun 8.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myelodysplastic syndromes (MDS) are hematological malignancies characterized by ineffective hematopoiesis and increased apoptosis in the bone marrow, which cause peripheral cytopenia. Mitochondria are key regulators of apoptosis and a site of iron accumulation that favors reactive oxygen species (ROS) production with detrimental effects on cell survival. Although the energy metabolism could represent an attractive therapeutic target, it was poorly investigated in MDS. The purpose of the study was to analyze how the presence of myelodysplastic hematopoiesis, iron overload and chelation impact on mitochondrial metabolism. We compared energy balance, OxPhos activity and efficiency, lactic dehydrogenase activity and lipid peroxidation in mononuclear cells (MNCs), isolated from 38 MDS patients and 79 healthy controls. Our data show that ATP/AMP ratio is reduced during aging and even more in MDS due to a decreased OxPhos activity associated with an increment of lipid peroxidation. Moreover, the lactate fermentation enhancement was observed in MDS and elderly subjects, probably as an attempt to restore the energy balance. The biochemical alterations of MNCs from MDS patients have been partially restored by the in vitro iron chelation, while only slight effects were observed in the age-matched control samples. By contrast, the addition of iron chelators on MNCs from young healthy subjects determined a decrement in the OxPhos efficiency and an increment of lactate fermentation and lipid peroxidation. In summary, MDS-MNCs display an altered energy metabolism associated with increased oxidative stress, due to iron accumulation. This condition could be partially restored by iron chelation.
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http://dx.doi.org/10.1038/s41598-020-66162-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280296PMC
June 2020

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

J Clin Med 2020 Jun 1;9(6). Epub 2020 Jun 1.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.
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http://dx.doi.org/10.3390/jcm9061670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355574PMC
June 2020

Novel Multiplex Droplet Digital PCR Assays to Monitor Minimal Residual Disease in Chronic Myeloid Leukemia Patients Showing Atypical Transcripts.

J Clin Med 2020 May 13;9(5). Epub 2020 May 13.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT-PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients' molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical patients, paving the foundation to include them in treatment-free remission protocols.
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http://dx.doi.org/10.3390/jcm9051457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290999PMC
May 2020
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