Anna Sicuranza

Genetic predisposition and induced pro-inflammatory/pro-oxidative status may play a role in increased atherothrombotic events in nilotinib treated chronic myeloid leukemia patients

Several reports described an increased risk of cardiovascular (CV) events, mainly atherothrombotic, in Chronic Myeloid Leukemia (CML) patients receiving nilotinib. However, the underlying mechanism remains elusive. The objective of the current cross-sectional retrospective study is to address a potential correlation between Tyrosine Kinase Inhibitors (TKIs) treatment and CV events. One hundred and 10 chronic phase CML patients in complete cytogenetic response during nilotinib or imatinib, were screened for CV events and evaluated for: traditional CV risk factors, pro/anti-inflammatory biochemical parameters and detrimental ORL1 gene polymorphisms (encoding for altered oxidized LDL receptor-1). Multivariate analysis of the whole cohort showed that the cluster of co-existing nilotinib treatment, dyslipidaemia and G allele of LOX-1 polymorphism was the only significant finding associated with CV events. Furthermore, multivariate analysis according to TKI treatment confirmed IVS4-14 G/G LOX-1 polymorphism as the strongest predictive factor for a higher incidence of CV events in nilotinib patients. Biochemical assessment showed an unbalanced pro-inflammatory cytokines network in nilotinib vs imatinib patients. Surprisingly, pre-existing traditional CV risk factors were not always predictive of CV events. We believe that in nilotinib patients an induced “inflammatory/oxidative status”, together with a genetic pro-atherothrombotic predisposition, may favour the increased incidence of CV events. Prospective studies focused on this issue are ongoing.

Molecular insight into the biology and clinical course of hairy cell leukemia utilizing immunoglobulin gene analysis

The B cell receptor (BCR) is the functional distinguishing unit that defines any B cell. Immunoglobulin gene (IG) status is preserved in the neoplastic B cell clone and can provide an indicator of the maturation stage reached by the B cell prior to transformation. In hairy cell leukemia (HCL), several pieces of data from IG analysis provide clear hints regarding the cell of origin and the ongoing selective interactions of the tumor BCR with environmental stimuli. HCLs have variable levels of IG somatic mutations, and continue somatic mutations at low levels as well as IG class switching after transformation. More recent data also show the occurrence of selective events in the light chain of the BCR, suggesting a dominant role for IG status in the pathogenesis of HCL. Moreover, it has recently emerged that an unmutated status of the HCL IG can be associated with failure to respond to cladribine, genetic abnormalities indicative of poor outcome, and aggressive disease. These observations suggest that IG analysis may have biological and prognostic relevance in HCL and merits further characterization.

Peptide vaccines for hematological malignancies: a missed promise?

Despite the crucial aid that newly developed target therapies are providing to chemotherapy and stem cell transplant, the cure for many hematological malignancies is still an unmet need. Although available therapies are able to induce an effective debulking of the tumor, most of the time, an insidious minimal residual disease survives current treatments and it is responsible for an immediate or delayed relapse. Peptide-derived antitumor vaccines have been developed with the idea that an artificially “educated” immune system may exert an active specific antitumor response able to control and ultimately eradicate underlying post-treatment residual disease. This review will summarize current knowledge of peptide vaccines for hematological malignancies, trying to analyze promises and pitfalls of a safe and intelligent tool that after many years from its first appearance has not yet established its potential role as alternative immune mediated therapeutic approach for hematopoietic tumors.

Insight into the behavior of hairy cell leukemia by immunogenetic analysis.

The B cell receptor (BCR) is the functional distinguishing unit that defines any B cell. Immunoglobulin gene (IG) status is preserved in the neoplastic B cell clone and can provide an indicator of the maturation stage reached by the B cell prior to transformation. In hairy cell leukemia (HCL), several data from IG analysis provide clear hints regarding the cell of origin and the ongoing selective interactions of the tumor BCR with environmental stimuli: HCLs (i) have variable levels of IG somatic mutations, (ii) continue somatic mutations at low levels, (iii) have active processes of IG class switching after transformation, and (iv) have a relatively high frequency of selective events in the light chain of the BCR. It has recently emerged that an unmutated status of the HCL-derived IG can be associated with failure to respond to cladribine, genetic abnormalities indicative of poor outcome, and aggressive disease. These observations suggest that IG gene analysis may have biological and prognostic relevance in HCL and merits further characterization.

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype‐switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour‐related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGKI/IGKII, IGKI/IGLI and IGLI/IGLII transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single‐cell analysis documented co‐expression of the tumour‐related IGLs in 5/6 cells (83%). In the IGK/IGL co‐expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour‐derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re‐induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery. Copyright

A pilot monocentric analysis of efficacy and safety of Fludarabine-Campath combination (Flucam) as first line treatment in elderly patients with chronic lymphocytic leukaemia and Tp53 disfunction

Chronic lymphocytic leukaemia (CLL) is a leukaemia of the elderly. Patients with CLL and TP53 disruption by 17p13 deletion (17pdel) and/or mutation (TP53 CLL) represent the group with the poorest outcome (Dohner et al, 2000; Forconi et al, 2008; Zenz et al, 2008; Rossi et al, 2009). The emergence of additional molecular aberrations, particularly after treatment, may cause further differential outcome (Forconi et al, 2008). Thus, an effective strategy upfront is still required for TP53 CLL. Alemtuzumab is a monoclonal antibody that induces cell death via TP53-independent mechanisms and is effective in TP53 CLL (Stilgenbauer & Dohner, 2002). Alemtuzumab may increase efficacy in combination with Fludarabine (FluCam) (Elter et al, 2005). However the role of FluCam in elderly patients with TP53 CLL has never been specifically investigated. We performed a pilot analysis of efficacy and toxicity of full dose FluCam in a consecutive series of ‘high risk’ elderly (>60 years) TP53 CLL patients requiring treatment for the first time (CLL1 group, n = 7), that received FluCam between 2005 and 2010 at our institution (Table I). The CLL1 patients were compared with a consecutive series of TP53 CLL with

Residual peripheral blood CD26+leukemic stem cells in chronic myeloid leukemia patients during TKI therapy and during treatment-free remission

Chronic myeloid leukemia (CML) patients in sustained “deep molecular response” may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38− LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38− stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/μL, range 0.27–698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012–0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006–0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that “circulating” CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a “stem cell response” threshold to achieve and maintain TFR are ongoing.

Study of gene polymorphisms as predictors of treatment efficacy and toxicity in patients with indolent non-Hodgkin lymphomas and mantle-cell lymphoma receiving bendamustine and rituximab

Bendamustine is used in combination with rituximab (BR) to treat indolent non‐Hodgkin lymphomas (iNHL) and mantle cell lymphoma (MCL). The variability in treatment efficacy and toxicity could be related to single nucleotide polymorphisms (SNPs) in immune response genes. We would like to show a correlation between SNPs and treatment outcome in iNHL and MCL patients receiving BR. We investigated some SNPs that had already been associated with NHL outcome. Samples were genotyped for the IL2 (rs2069762), IL10 (rs1800890, rs10494879), VEGFA (rs3025039), IL8 (rs4073), CFH (rs1065489) and MTHFR (rs1801131) SNPs by allelic discrimination assays. We enrolled 70 patients that received rituximab 375 mg/m2 and bendamustine 90 mg/m2 every 28 days, both as first‐line treatment and ≥ second‐line regimens. Overall response rate was 97·1% (complete response [CR] rate 73·9%). Treatment toxicity included grade 3–4 neutropenia (24/70 patients), infections (21/70 patients; 1/70 grade 3), skin rash (26/70 patients; 2/70 grade 3). After a median follow‐up of 24 months we did find any correlation between the analysed SNPs, CR rate and PFS. However, we demonstrated an association between the SNP in IL2 (rs2069762) and the onset of skin rash (P = 0·0001). Our study suggests a role for cytokine SNPs in bendamustine‐related toxicity, which could represent a promising research field.

Cardiovascular toxicity in patients with chronic myeloid leukemia treated with second-generation tyrosine kinase inhibitors in the real-life practice: Identification of risk factors and the role of prophylaxis

To the Editor: Long-term treatment with the second-generation tyrosine kinase inhibitors (2TKIs) nilotinib and dasatinib may result in cardiovascular (CV) complications. Accumulating evidence suggests that the combination of a median age at the time of chronic myeloid leukemia (CML) diagnosis of greater than 60 years, when CV adverse events (AEs) are common, and the CV toxicity of 2TKIs represents per se a potential predisposing factor, which requires preventive strategies and CV surveillance in patients with CML. Previous studies have suggested the usefulness of the systematic coronary risk evaluation (SCORE) assessment at disease baseline, a 10-year risk estimation of fatal CV disease based on sex, age, smoking habits, systolic blood pressure, and total cholesterol levels, to identify patients who are at heightened risk of CV AEs during nilotinib treatment. A preventive strategy with primary prophylaxis based on aspirin remains under discussion. We therefore analyzed a large real-life cohort of Italian patients with CML treated with a 2TKIs as firstor subsequent-line of treatment. The primary objective was to evaluate the incidence of CV AEs and the association with the SCORE assessment and other baseline risk factors. The secondary objectives were to evaluate the role of primary prophylaxis in preventing CV atherothrombotic events. We identified consecutive adult patients with CML who initiated nilotinib or dasatinib as firstor subsequent-line treatment, between January 2012 and December 2015 in 20 Italian centers. Patients were stratified into low-moderate (SCORE 5%) or high-very high (SCORE >5%) CV risk. Additional risk factors were the presence of diabetes, body mass index>24.5 kg/m, mild or severe renal insufficiency, and dyslipidemia. Patients were also evaluated for comorbidities and a positive anamnesis of CV diseases, including angina, myocardial infarction, stroke, heart failure, arterial hypertension, cardiomyopathy, heart arrhythmia, valvular heart disease, aortic aneurysms, ischemic cerebrovascular events, peripheral artery disease, thromboembolic disease, and venous thrombosis. The presence of antithrombotic prophylaxis before initiating CML treatment was also recorded. The probability of the cumulative incidence of CV and atherothrombotic AEs was estimated after initiating treatment with 2TKIs. The cumulative incidence of deep molecular response (MR) was evaluated from the initiation of 2TKIs treatment. Multivariate analyses were performed using the Cox proportional hazards regression model. A total of 506 patients with CML were retrospectively recruited. The patients’ characteristics are shown in Supporting Information Table S1. The mean age at diagnosis was 52 years (range 18–87) and 57% were men. Sokal score was intermediate-high in 55% of patients. The mean follow-up time since CML diagnosis was 5.4 years (range 0.2–23). Overall, 286 patients were treated with nilotinib and 220 with dasatinib. 2TKIs were administered as first-, second-, and third-line treatment in 61%, 32%, and 7% of cases, respectively. The reasons for switching treatments in 196 patients were inefficacy in 63.8%, intolerance in 29.6%, and protocol requirements in 6.6%. The majority of patients (93%) were classified as at low-intermediate risk (SCORE 5%) and 7% as at high-very high risk (SCORE>5%). A positive history for CV diseases was noted in 181 (35.8%) patients. The 60-month CV AE cumulative incidence registered in the total cohort of patients was 21.762.8%. Patients treated with nilotinib and dasatinib showed CV AE incidence of 24.763.9% and 16.463.7%, respectively (P5 .25; NS) (Supporting Information Figure S1). Patients treated with 2TKIs administered as firstor second-line of treatment and as subsequent-line treatment showed a CV AE incidence of 12.963.5% and 22.964.4%, respectively (P5 .004). Patients with high-very high SCORE showed significantly high incidence of CV AEs (46.6616.6% vs. 2062.8%; P< .001). The mean time between the initiation of 2TKI treatment and the occurrence of CV AEs was 35.5 (range 1–69) months. Overall, 68 CV AEs were registered, with 2 event-related deaths; 40% of CV AEs were graded as 3/4 of common toxicity criteria. Supporting Information Table S2 reports the CV AEs and their management in the reallife. We did not find any association between TKI dose and CV AE incidence. The frequency of peripheral arterial disease (PAOD or atheromasic carotid disease) was significantly high in patients undergoing nilotinib treatment. Two patients died due to myocardial infarction during treatment. Overall, in 44% of cases 2TKI treatment did not require dose modification; 16% of patients reduced the dose and

GENE POLYMORPHISMS AS PREDICTORS OF TREATMENT EFFICACY AND TOXICITY IN PATIENTS WITH INDOLENT NON-HODGKIN LYMPHOMAS RECEIVING BENDAMUSTINE AND RITUXIMAB

chemoimmunotherapy. Progression‐free survival (PFS) and global survival (OS) rates were estimated using the method of Kaplan–Meier. Univariate and multiple Cox regression models were used to assess the effect of covariates on PFS and OS. Results: Median (range) age at diagnosis was 62 years (32–83). Forty‐eight percent of patients had high‐risk FLIPI and 36.7% were high‐risk FLIPI‐2. Patient characteristics are shown in the Table 1. The most commonly front‐line therapies administered were R‐CHOP (56.9%) and R‐CVP (40%). Rituximab maintenance was administered to 48 patients (73.8%). Overall, 67.2% of patients achieved complete remission (CR) and 12.5% partial remission (PR) with no significant differences between both regimens. After a median follow‐up of 60 months (range, 0–185), 67.7% of patients are still in CR, 15.4% have relapsed, and 23.1% have died. Neither the median PFS nor the median OS were reached. The estimated 5‐year PFS and OS among all patients were 67.4% and 83.4%, respectively. Variables influencing PFS and OS in the univariate analysis were age > 60 years (p = 0.02), Hb <12 g/dL (p = 0.036), raised serum LDH (p = 0.001), albumin <3 mg/dL (p = 0.029), high‐risk FLIPI (p = 0.01), high‐risk FLIPI‐2 (p = 0.001), not receive Rituximab maintenance (p = 0.04), disease progression <2 years after diagnosis (p < 0.0001), and disease refractoriness (p < 0.0001). Multivariate analysis confirmed raised serum LDH (p = 0.034) and refractoriness to front‐line therapy (p = 0.021) as the two variables affecting OS while disease progression <2 years after diagnosis was the only variable affecting PFS (p = 0.007). Conclusions: Our results show that refractoriness to chemoimmunotherapy and early progression after treatment were the most relevant variables affecting outcome of patients. Both factors should be used to stratify the risk of patients with FL. Likewise, we did not found any OS and PFS advantage among patients receiving maintenance. UNL, upper normal limit.