Circulating cell-free DNA for target quantification in hematologic malignancies: Validation of a protocol to overcome pre-analytical biases.

Circulating tumor DNA (ctDNA) has become the most investigated analyte in blood. It is shed from the tumor into the circulation and represents a subset of the total cell-free DNA (cfDNA) pool released into the peripheral blood. In order to define if ctDNA could represent a useful tool to monitor hematologic malignancies, we analyzed 81 plasma samples from patients affected by different diseases. The results showed that: (i) the comparison between two different extraction methods Qiagen (Hilden, Germany) and Promega (Madison, WI) showed no significant differences in cfDNA yield, though the first recovered higher amounts of larger DNA fragments; (ii) cfDNA concentrations showed a notable inter-patient variability and differed among diseases: acute lymphoblastic leukemia and chronic myeloid leukemia released higher amounts of cfDNA than chronic lymphocytic leukemia, and diffuse large B-cell lymphoma released higher cfDNA quantities than localized and advanced follicular lymphoma; (iii) focusing on the tumor fraction of cfDNA, the quantity of ctDNA released was insufficient for an adequate target quantification for minimal residual disease monitoring; (iv) an amplification system proved to be free of analytical biases and efficient in increasing ctDNA amounts at diagnosis and in follow-up samples as shown by droplet digital PCR target quantification. The protocol has been validated by quality control rounds involving external laboratories. To conclusively document the feasibility of a ctDNA-based monitoring of patients with hematologic malignancies, more post-treatment samples need to be evaluated. This will open new possibilities for ctDNA use in the clinical practice.

Real-life comparison of nilotinib versus dasatinib as second-line therapy in chronic phase chronic myeloid leukemia patients.

Tyrosine kinase inhibitors (TKIs), the backbone of treatment for chronic phase chronic myeloid leukemia patients (CP-CML), have changed the long-term outcome of the disease. Nonetheless, over 20% of patients fail front-line therapy due to intolerance or resistance. A head-to-head comparison of dasatinib and nilotinib as second-line treatment outside of sponsored clinical trials has not been reported. We retrospectively analyzed 131 CP-CML patients who, after front-line imatinib failure, switched to a second-line therapy with nilotinib (59, 45%) or dasatinib (72, 55%). Median duration of second-line treatment was 33 months (range 2-100). The reason for switching therapy was resistance in 83.2% and intolerance in 16.8% of patients. The overall survival of the entire cohort at 7 years was 78.9%, while it was 72% and 85.6% for patients treated with dasatinib and nilotinib, respectively (p=0.287). With regard to efficacy after 12 months of treatment, 108 patients were evaluable for molecular response: 47% achieved a major molecular response and 18.2% a deep molecular response with dasatinib, compared to 38% and 16.2% with nilotinib (p=ns). We observed 35% of grade 3-4 adverse events, more frequently in the dasatinib group (47%) compared to the nilotinib group (22%), without affecting molecular responses. Our study suggests that, in the real-life setting, dasatinib and nilotinib used as second-line treatment in CP-CML are equally effective, with high molecular response rates and an acceptable tolerability.

Acute promyelocytic leukemia (APL) in very old patients: real-life behind protocols.

BACKGROUND: Acute promyelocytic leukemia (APL) is uncommon among subjects aged ≥ 70 years and the better therapeutic strategy represents an unmet clinical need. MATERIALS AND METHODS: This prompted us to explore our real-life data on a retrospective cohort of 45 older APL patients (≥ 70 years) consecutively diagnosed at eight different hematologic institutions in Latium, Italy, from July 1991 to May 2019. RESULTS: Two patients (4.4%) died from early hemorrhagic complications before treatment could begin. Twenty-two patients (51.1%) (Group A) were enrolled or treated according to standard clinical protocols, while 21 (48.8%) (Group B) received an ATRA-based personalized approach due to poor performance status. Morphologic complete remission (CR) after induction therapy was achieved in 33 patients (76.7%) with 100% of patients in Group A and 52.3% in Group B (p < 0.001). Molecular CR was documented in 30 patients (69.7%) [20/22 (90.9%) in Group A and 10/21 (47.6%) in Group B (p = 0.002)]. Ten patients (23.2%) died during induction therapy, all in Group B. Five-year overall survival (OS) of the entire cohort was 46.1% (95% CI 28.2-64.0), with 72.6% (95% CI 42.9-100) in Group A vs. 27.2% (95% CI 7.5-46.9) in the Group B (p = 0.001). CONCLUSIONS: The present analysis highlights that almost half of the patients received sub-optimal induction treatments and registered dismal outcomes demonstrating the importance of adopting standard therapies instead of modified or reduced personalized approaches also in the setting of frail older patients.

Long term follow-up of frontline Dasatinib in older patients with chronic myeloid leukemia in chronic phase treated outside clinical trials: a real-life cohort observational study.

BACKGROUND: A limited amount of data has been published in chronic-phase chronic myeloid leukemia (CP-CML) patients aged >75 years treated frontline with second-generation tyrosine kinase inhibitors. AIMS: To address this issue in a clinical 'real-life' setting, we retrospectively analyzed 45 CP-CML patients (pts) followed in 20 Italian Centers and treated frontline with dasatinib (DAS). PATIENTS AND METHODS: Median age was 78.4 years (range 75-89.2 years). DAS starting dose was 100 mg QD in 35 pts (77.7%), 80 mg QD in 1 pts (2.2%) and 50 mg QD in 9 pts (20.1%), respectively. The median follow-up was 42.6 months (IQR 20.4 - 63.3). RESULTS: Grade 3 and 4 side effects, both hematological and non-hematological, were detected in 6 (13.3%) and 12 (26.6%) pts, respectively. Pleural effusions of all grades occurred in 13 pts (28.8%) after a median period of DAS exposure of 14.7 months (IQR 3.0 - 33.1). The rates of DAS dose reduction and permanent drug discontinuation were 53.3% and 20.0%, respectively. As the best response, 42/45 patients (93.3%) achieved a complete cytogenetic response (CCyR), 35/45 (77.7%) a major molecular response (MMR) and 24/45 (53.3%) a deep molecular response (both MR 4.0 and MR 4.5). Only 1 patient (2.2%) progressed to the blast phase after 13 months of therapy; 8 deaths were observed (1 CML-related and 7 CML-unrelated). Cumulative event-free survival and overall survival at 36 months were 64.7% (95%, CI 49.4 - 80.0) and 82.3% (95%, CI 70.3-94.3), respectively. CONCLUSION: These findings, although evaluated in a limited and selected cohort of patients, suggest that DAS might be effective in older patients (aged >75 years) affected by CP-CML with acceptable toxicity.

Predictive factors for response and survival in elderly acute myeloid leukemia patients treated with hypomethylating agents: a real-life experience.

Predictive factors of response to hypomethylating agents (HMA) in elderly acute myeloid leukemia (AML) patients remain unclear in the real-life setting and no direct comparison between azacitidine (AZA) and decitabine (DEC) has been carried out. We retrospectively evaluated 110 AML patients treated with HMA (78 AZA, 32 DEC) as first-line therapy outside of clinical trials. Median age was 75 years (range 58-87). The median overall survival (OS) of the entire cohort was 8.0 months (95% CI 6.1-10), without significant differences among the subgroups: AZA 8.8 months vs DEC 6.3 months (p = 0.291). HMA treatment yielded an overall response rate (ORR) of 40% (AZA 37% vs DEC 47%, p = 0.237). A stable disease (SD) after 4 HMA cycles was not associated with a worse survival outcome compared with an early optimal response. Factors independently associated with a better OS were transfusion independence during treatment (p = 0.049), achievement of an optimal response to treatment (p < 0.001), and a baseline hemoglobin level ≥ 9.25 (p = 0.018). A bone marrow (BM) blast count ≥ 30% (p < 0.001) and a therapy-related AML (p = 0.008) remain poor survival predictors. Of the available biologic features, an adverse risk category according to the ELN classification was significantly associated with a shorter survival over the intermediate risk category (p = 0.034). Disease progression remains the primary cause of death. Infectious complications were more severe (p = 0.036) and occurred earlier (p = 0.006) in the DEC group compared with that of the AZA group. In conclusion, clinical prognostic factors associated to response and survival have been identified without significant associations concerning overall outcomes between the two HMAs.

Switch from branded to generic imatinib: impact on molecular responses and safety in chronic-phase chronic myeloid leukemia patients.

Since July 2017, different generic imatinib formulations have been introduced in Italy for the treatment of patients with chronic myeloid leukemia (CML). We analyzed 168 chronic phase CML patients treated with branded imatinib for a median of 12 years (range 1-16) at a single institution who switched to a single generic formulation in order to assess the safety and impact on molecular response. The Sokal risk was low/intermediate/high in 63%, 33%, and 4% of patients, respectively. The median duration of generic imatinib treatment was 19 months (range 4-22). Twenty-seven percent of patients were in MMR and 73% were in deep molecular responses (MR4-4.5) at the time of the switch. After 12 months of treatment with generic imatinib, 140 patients were evaluable for response: 23.6% and 76.4% were respectively in MMR and in deep molecular response. When the degree of response was compared with the best molecular response observed with branded imatinib, it was found that 84% of patients maintained the response previously achieved, 6% improved it, and 10% of patients had a molecular fluctuation from the previous deep molecular response to MMR. Only 1 patient lost the MMR and no patient switched to another TKI for inefficacy. In terms of safety, 20% of patients reported new or worsening side effects, but only 2 patients returned to branded imatinib for toxicity. Our data show that the switch to generic imatinib in patients who have been previously treated with branded imatinib appears to maintain efficacy, although a proportion of patients experience new or worsening side effects.

Identification of predictive factors for overall survival at baseline and during azacitidine treatment in high-risk myelodysplastic syndrome patients treated in the clinical practice.

The outcome of high-risk myelodysplastic syndrome (MDS) patients treated with 5-azacitidine (5-AZA) in the real-life setting remains largely unknown. We evaluated 110 MDS patients (IPSS intermediate 2/high) treated outside of clinical trials at a single institution between September 2003 and January 2017. Median duration of therapy was 9.5 cycles. The overall survival (OS) of the whole cohort was 66.1% at 1 year and 38.3% at 2 years. No differences in terms of OS were observed with regard to gender (p = 0.622) and age at baseline (< 65 years, 65-75, and > 75 years, p = 0.075). According to the IPSS-R, the very high-risk group had an inferior 2-year OS (17%) compared with intermediate-group patients (64%, p < 0.001). Transfusion independency at baseline was identified as a favorable prognostic factor on 1-year (66.8%) and 2-year OS (43.4%) (p < 0.001). After four cycles, the persistence of bone marrow blasts > 10% identified patients with a worse outcome, with a 2-year OS of 9.4% (p = 0.002). The occurrence of an infection during the first four cycles impacted on the 2-year OS (31.6% vs 58.3% in patients without infections, p = 0.032). Patients receiving at least 24 cycles of the drug have a 5-year OS of 38.2%. This analysis allowed to identify features at baseline or during treatment with 5-AZA associated with a different 2-year OS.

Ten-year outcome of chronic-phase chronic myeloid leukemia patients treated with imatinib in real life.

Imatinib, the first BCR/ABL kinase inhibitor approved for the treatment of chronic myeloid leukemia (CML), has changed the long-term outcome of patients affected by this disease. The aim of our analysis was to report, after a median follow-up of 10.2 years (range 5.8-14.8), the long-term outcome, efficacy, and safety of imatinib treatment (frontline and after interferon failure) in a single institution cohort of 459 patients with CML in chronic phase treated outside of clinical trials. The 10-year overall survival of the whole cohort was 77.1%, while the 10-year probability of dying due to CML and other causes was 7.8% and 16%, respectively. The prognostic value of the BCR-ABL1 ratio at 3 months (⩽ 10%) and of complete cytogenetic response and major molecular response at 1 year was confirmed also in the real-life practice. The EUTOS long-term survival score better stratified the baseline risk of dying of CML compared with other risk scores. Two hundred thirty-six (51.4%) patients achieved a deep molecular response during imatinib treatment after a median time of 4.57 years, and 95 (20.6%) had a stable deep molecular response maintained for at least 2 consecutive years. Imatinib was associated with a low rate of serious cardiovascular events and second neoplasia. This 10-year real-life follow-up study shows that imatinib maintains efficacy over time and that long-term administration of imatinib is not associated with notable cumulative or late toxic effects.

Changes in estimated glomerular filtration rate in chronic myeloid leukemia patients treated front line with available TKIs and correlation with cardiovascular events.

We investigated the median estimated glomerular filtration rate (eGFR) changes in chronic myeloid leukemia (CML) patients treated front line with tyrosine kinase inhibitors (TKIs). A large cohort of 397 patients-320 treated front line with imatinib, 25 with dasatinib, and 53 with nilotinib-was retrospectively analyzed at a single institution. The eGFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration equation for all patients at baseline and then at 6 and 12 months, and at the last follow-up. Taking into account eGFR changes during the first year of treatment and excluding other possible cardiovascular risk factors, we considered also the percentage of cardiovascular events in patients with modifications of this single parameter. Imatinib induced a decrease in median eGFR (p = 0.01): 42 patients treated with imatinib had a cardiovascular event, related to modification of eGFR, in the absence of other cardiovascular risk factors. In patients treated with nilotinib, the median eGFR did not decline from baseline: only 1 patient experienced an ischemic event, but the eGFR remained unchanged. In patients treated with dasatinib, the mean eGFR did not change significantly: 3 patients experienced a cardiac ischemic event, but in all patients the eGFR remained unchanged over time, while advanced age and metabolic alterations contributed to the ischemic events. This long-term follow-up has documented that imatinib may induce changes in the eGFR, which may contribute to the onset of ischemic events. Further analyses on larger series of CML patients are required to conclusively define the potential renal toxicity of second generation TKIs and the consequent risk of developing ischemic events.

Clinical results according to age in patients with chronic myeloid leukemia receiving imatinib frontline: The younger, the later, the worse?

OBJECTIVES: To evaluate differences in clinical results according to age among patients with chronic myeloid leukemia (CML). METHODS: 207 consecutive CML patients treated with imatinib frontline were revised, dividing them in young adults (>20 < 45 years) (YA), middle-aged adults (≥45 < 65 years) (MA) and elderly (≥65 years) (EL). RESULTS: Cumulative incidence of complete cytogenetic response (CCyR) and major molecular response (MMolR) were significantly higher in MA compared with YA and EL (P < .001 for CCyR and P = .001 for MMolR). Number of total events was lower in MA (8 [11.1%] vs 21 [34.4%] in YA and 28 [37.8%] in EL, P = .001): no difference was observed for blastic evolution (P = .478). Number of deaths was higher in the EL (12 [16.2%] vs 2 [3.2%] in YA and 0 in MA, P < .001): however, 11/12 deaths in EL were not related to CML. The PFS curve in MA was significantly longer than in YA and in EL (P = .02). The OS curve in EL was significantly shorter than in YA and in MA (P < .001). CONCLUSIONS: Age at diagnosis influences significantly the course of CML patients treated with imatinib: a possible explanation of the counterintuitive worse course in YA is the delayed diagnosis compared to elderly.

Independent prognostic impact of CD15 on complete remission achievement in patients with acute myeloid leukemia.

The prognostic role of CD15 in acute myeloid leukemia (AML) has been tested in different studies with conflicting results. To address this issue, we retrospectively evaluated a cohort of 460 AML patients of all ages with the exclusion of acute promyelocytic leukemia (M/F 243/217, median age 50.6 years [range 0.9-81.2]) intensively treated at our institute between January 1999 and December 2010. CD15 positivity was found in 171 of 406 evaluable patients (42.1%). Complete remission (CR) was achieved by 334 patients (72.6%), while 82 (17.8%) were resistant and 44 (9.6%) died during induction: the median CR duration was 15.5 months (range 0.6-176.0), with 2-year disease-free survival rate of 45.1% (95% confidence interval 39.6-50.6). The median overall survival was 14.4 months (range 0.3-177.0), with 2-year overall survival rate of 42.2% (95% confidence interval 37.5-46.9). At univariate analysis for CR achievement, age < 60 years (P < .001), World Health Organization classification (P = .045), low-risk karyotype (P < .001), no high-risk karyotype (P = .006), positivity for AML-ETO (P = .004)/CBFß-MYH11 (P = .003)/CD15 (P = .006)/CD11b (P = .013), negativity for FLT3-ITD (P = .001), Hb > 8 g/dL (P = .020), and white blood cell < 50 × 109 /L (P = .034) had a favorable impact. At a multivariate logistic regression model, CD15 positivity (P = .002), age < 60 years (P = .008), white blood cell < 50 × 109 /L (P = .017), and low-risk/no high-risk karyotype (P = .026/P = .025) retained an independent prognostic role on CR achievement. The baseline assessment of CD15 positivity appears to have a role in the risk evaluation for CR achievement in AML patients undergoing intensive chemotherapy and should be assessed in prospective studies together with other clinical and biologic features already reported.

Ruxolitinib in clinical practice for primary and secondary myelofibrosis: an analysis of safety and efficacy of Gruppo Laziale of Ph-negative MPN.

Ruxolitinib, a JAK1 and JAK2 inhibitor, has been tested and approved for the treatment of primary and secondary myelofibrosis (MF). Aim of our study is to report safety and efficacy of ruxolitinib in 98 patients affected by MF treated outside clinical trials and collected and treated consecutively by the Lazio Cooperative Group for Ph negative myeloproliferative diseases.There were 45 males and 53 females; median age was 61.8 years (range 35.3-88). Forty-five patients were diagnosed as primary MF and 53 as secondary MF. Seventy-seven patients (78.5%) experienced constitutional symptoms at baseline, and out of 94 patients tested, 66 (70%) were JAK2V617F mutated. Overall, 40 patients received hydroxyurea as firstline treatment, 30 patients received other chemotherapeutic approaches, whereas 28 were treated with ruxolitinib frontline. Median time from diagnosis to start of ruxolitinib in the whole cohort was 34.6 months. Fifty-eight patients (59%) required a dose reduction during the first 3 months due to hematological toxicity in the majority of cases. At 48 weeks, 52% of patients obtained a clinical benefit: of them 7 patients (7%) had a CR, 10 (10%) a PR, 6 patients (6%) a CI, and 28 patients (28.5%) a spleen response. Overall, 66% of patients had disappearance of baseline symptoms burden. After 1 year, of 72 evaluable patients, 52% achieved and maintained a clinical benefit. Adverse events of special interest at any grade included anemia (39.7%), thrombocytopenia (25.5%), infections (16.3%, of which 10 were bronchopneumonia), fluid retention (3%), diarrhea (2%) and abdominal pain (2%). After a median follow-up of 16 months from start of ruxolitinib, median daily dose decreased to 10 mg BID and 21 patients (21%) discontinued the drug. The results of this retrospective multicentric analysis confirmed the efficacy of ruxolitinib outside clinical trials with more than half of treated patients achieving and maintaining a clinical benefit and most of them reporting relief from symptoms.

Risk factors for infections in myelofibrosis: role of disease status and treatment. A multicenter study of 507 patients.

Although infectious complications represent a relevant cause of morbidity and mortality in patients with myelofibrosis (MF), little is known about their incidence, outcome and risk factors. We retrospectively evaluated a cohort of 507 MF patients, diagnosed between 1980 and 2014 in five Italian hematology centers, to define the epidemiology of infections and describe the impact of ruxolitinib (RUX) treatment. Overall, 112 patients (22%) experienced 160 infectious events (grade 3-4, 45%) for an incidence rate of 3.9% per patient-year. Infections were mainly bacterial (78%) and involving the respiratory tract (52% of cases). Also, viral (11%) and fungal infections (2%) were recorded. Overall, infections were fatal in 9% of the cases. Among baseline features, high/intermediate-2 IPSS category (HR 1.8, 95%CI:1.2-2.7; P = 0.02) and spleen length ≥10 cm below left costal margin (HR 1.6, 95%CI:1.1-2.5; P = 0.04) were associated with higher infectious risk in multivariate analysis. Overall, the rate of infections was higher in the cohort of 128 RUX-treated patients (44% vs. 20%, P < 0.001). In conclusion, IPSS-category and splenomegaly, emerged as the main risk factors for infections in MF. RUX-treated patients experienced significantly more infection episodes; however, future prospective studies are needed to isolate the confounding contribution of other risk factors such as disease stage. Am. J. Hematol. 92:37-41, 2017. © 2016 Wiley Periodicals, Inc.

Acute promyelocytic leukemia in patients aged >70 years: the cure beyond the age.

All-trans retinoic acid (ATRA) has made acute promyelocytic leukemia (APL) a very curable disease also in patients aged >60 years; however, there are only few case reports in very elderly APL patients. To address this issue, we reviewed treatment results in 13 patients aged >70 years with newly diagnosed APL followed at our institution from January 1991 to December 2008. According to Sanz score, seven patients were at low risk, five at intermediate risk, and one at high risk. Induction therapy consisted of ATRA + idarubicin in nine patients (3/9 with reduced idarubicin dosage) and ATRA alone in four patients; in this latter group, however, 2/4 needed to add chemotherapy (CHT) due to hyperleukocytosis during ATRA treatment. All patients achieved both morphological and molecular complete remission (CR) after a median time of 51 [interquartile range (IR) 43-55] and 114 (IR 74-155) days, respectively. Infective complications were observed in 10/13 patients, APL differentiation syndrome in 3/13 patients. Twelve patients received consolidation therapy, followed by maintenance treatment in nine patients. Five patients relapsed after 7, 8, 11, 35, and 56 months. At present, seven patients are still alive, five died due to disease progression (four) or senectus while in CR (one), and one was lost to follow-up while in CR. The 5-year event-free survival was 56.1 % (95 % CI, 26.0-86.2); the 5-year overall survival (OS) was 64.5 % (95 % CI, 35.6-93.4). ATRA-based treatment of APL is safe and effective also in very elderly patients, with long-lasting disease-free OS.

Negative prognostic value of CD34 antigen also if expressed on a small population of acute promyelocytic leukemia cells.

Potential clinical significance of CD34 expression in acute promyelocitic leukemia (APL) has not been deeply investigated. We hereby analyzed the clinico-biological features and treatment outcome of APL patients in relation to CD34 expression, even when expressed in a small subpopulation: 114 APL patients homogeneously treated with the AIDA schedule were included in the study and prognostic correlation with respect to CD34 expression, both when expressed in association with CD2 and as isolated expression (cutoff ≥2 to <10 % or ≥10 %), were investigated. CD34 was associated to CD2 in 30 patients and was isolated in 19 patients. When compared to the CD34-negative population, CD34/CD2 expression identified a subgroup with characteristic features: M3 variant subtype (26 vs 7 % in the negative group, p = 0.02), bcr3 transcript subtype (73 vs 32 %, p = 0.001), high risk according to the risk of relapse (66 vs 17 %, p = 0.002), high incidence of differentiation syndrome (26 vs 12 %, p = 0.01), lower overall survival (88 vs 95 %), and a significantly higher rate of relapse (22 vs 13.8 %, p = 0.05). We then evaluated the prognostic value of isolated CD34 expression: it was detected in nine patients with a cutoff of expression ≥10 % and in 10 patients with a cutoff ≥2 but <10 %. Isolated CD34 positivity identified a subgroup with a classic morphology (79 %), bcr1 prevalence (53 %), higher rate of relapse (37 vs 13.8 % in the negative group, p = 0.002), higher incidence of differentiation syndrome (55 vs 12 %, p = 0.03), and lower overall survival (60 vs 95 %, p = 0.001). The results of our study confirm that CD34/CD2 expression characterizes a subset of APL with a high WBC count and a variant morphological subtype, associated with an unfavorable clinical course. We also show that the isolated expression of CD34, even at a low cutoff, identifies a group of classic APL with a negative prognosis. Further studies aimed at identifying other molecular signatures in CD34-positive patients are needed in order to optimize the therapeutic strategy for this subset of patients.