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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.

Background Limited information is available regarding the rate of long-term cardiovascular (CV) mortality in chronic myeloid leukaemia (CML) patients treated with second- and third-generation tyrosine kinase inhibitors (2ndG/3rdG TKIs) in the real-life practice. Methods We identified 656 consecutive CML patients treated with nilotinib, dasatinib, bosutinib and ponatinib. Results The 15-year CV-mortality free survival was 93 ± 2.8%. Age ≥65 years (p = 0.005) and a positive history of CV disease (p = 0.04) were significantly associated with a lower CV-mortality free survival. CV disease accounted for 16.5% and 5% of potential years of life lost (PYLL) in male and female patients, respectively. The standard mortality ratio (SMR) following ischemic heart disease (IHD) was 3.9 in males and 3.8 in female patients, meaning an excess of IHD deaths observed, in comparison with the population of control. Conclusion. Prevention strategies based on CV risk factors, in particular in those patients with a previous history of CV disease, should be considered.

Background Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure. Methods Thiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs. Results Here we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with β-catenin enables β-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone. Conclusions Our conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML. Electronic supplementary material The online version of this article (10.1186/s13046-019-1197-9) contains supplementary material, which is available to authorized users.

Take home messages Five tyrosine kinase inhibitors are available, the treatment strategy is still challenging. Baseline risk, comorbidities, and patient and physician expectations play a pivotal role. Treatment-free remission is a new opportunity.

Very elderly (> 75years) 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 78.5years 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 45.0months, 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 ≤80years and > 80years, respectively (p = 0.098). Five years OS was 75.7% and 61.6% in patients ≤80years and > 80years, 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.

Although once considered quite a rare condition in the past, female urethral diverticulum (UD) would now appear to have a higher frequency, perhaps due to greater attention from physicians. To date, there is no agreement on which is the best method for diagnosis of female UD. Traditionally, the approach was based on quite invasive techniques, such as voiding cystourethrography, and double-balloon urethrography, with satisfactory results but relevant limitations. More recent high-resolution imaging techniques, such as 2D-3D ultrasonography (US) and magnetic resonance imaging (MRI) have also been applied in the study of the abnormalities of the female urethra. US had the advantage of the outpatient setting, non-invasiveness and absence of contrast medium use; MRI, is characterized by high sensitivity thanks to multiplanar capability, with an optimal characterization of periurethral diseases or its abnormalities, and lack of ionizing radiation. A real innovation is represented by computer tomography-voiding cystourethrography, a rapid technique that allows for high quality simultaneous 2D and 3D images of the urethra, well correlated to MRI and consequently with surgical results. Here, we report and comment the available tools in the diagnosis of female UD, focusing particularly on pros and contra of different methods.

Arterial occlusive events (AOEs) represent emerging complications in chronic myeloid leukemia (CML) patients treated with ponatinib. We identified 85 consecutive CML adult patients who were treated with ponatinib in 17 Italian centers. Patients were stratified according to the Systematic Coronary Risk Evaluation (SCORE) assessment, based on sex, age, smoking habits, systolic blood pressure, and total cholesterol levels. The 60-month cumulative incidence rate of AOEs excluding hypertension was 25.7%. Hypertension was reported in 14.1% of patients. The median time of exposure to ponatinib was 28months (range, 3-69months). Patients with a high to very high SCORE risk showed a significantly higher incidence rate of AOEs (74.3% vs 15.2%, P&nbsp

The apical prolapse has always been considered the most complex of the defects of the pelvic floor, for both the difficulty of the surgical corrective technique and for the high post-surgical recurrence rate. Today, the laparoscopic sacrocolpopexy can be considered the standard treatment for apical prolapse. In the last years, several author performed robotic sacrocolpopexy, obtaining positive results. So, we developed a case-control study in order to compare the surgical outcome of robotic group with a control group of laparoscopic approach in patients with symptomatic apical pro-lapsed between January 2015 and December 2015 at University Hospital Policlinico “P. Giaccone” and Ospedali Riuniti “Villa Sofia-Cervello”, Palermo. Our experience shows that robotic sacrocolpopexy can be considered in positive way for clinical results obtained: all procedures were executed with no complications, we noted a lower intraoperative blood loss and a shorter hospital stay than in laparoscopic group. Although the mean operative time and the economic costs are higher in robotic surgery, this study demonstrates that the use of robotic platform for repairing of symptomatic apical vaginal prolapse is feasible, safe and associated with short-term satisfactory results, representing therefore a valid alternative to laparoscopic approach.

To evaluate the role of vaginal fractional CO2 laser treatment in the relief of Overactive Bladder (OAB) symptoms in post-menopausal women.Post-menopausal women who complained of one or more symptoms related to vulvo-vaginal atrophy (VVA), who experienced symptoms of OAB and who underwent vaginal treatment with fractional CO2 laser were enrolled in the study. At baseline (T0) and 30 days post-treatment T1), vaginal status (using Vaginal Health Index - VHI), subjective intensity of VVA symptoms (using a visual analog scale - VAS) and micturition diary were evaluated. OAB symptoms were also assessed using a validated questionnaire.Thirty patients were enrolled. A statistically significant improvement in VVA symptoms was observed and in VHI at T1 (p0.0001). A significant improvement was also identified in the micturition diary, in number of urge episodes and OAB-q (p0.0001). Nine of the 30 patients suffered from incontinence episodes and had improved at T1.We showed that fractionated CO2 laser vaginal treatment has proved to be effective in improving OAB symptoms in post-menopausal women. Moreover, it is a safe and efficacious measure for the relief of VVA related conditions. Further long-term studies are needed to confirm these preliminary results.

Purpose To test the efficacy, duration and safety of 100 U of botulinum toxin type A (BoNT/A) in women affected by idiopathic detrusor overactivity (IDO) and the effectiveness of subsequent injections. Methods In this double centre, prospective study con- ducted from March 2008 to March 2010, we selected women affected by IDO who failed to respond to various antimuscarinic agents, reported intolerable anticholinergic side-effects or contraindication to their use, also without any response to tibial stimulation. Medical history, physi- cal examination, standard urodynamic examination, uri- nalysis, urine culture, a 4-day voiding diary and a quality of life questionnaire were requested for all patients. A total amount of 100 U of BoNT/A were injected into the detrusor muscle. A second injection of BoNT/A was sug- gested to patients who experienced a relapse of initially improved symptoms. Results We enrolled a total number of 68 women. All patients showed significant improvement in urodynamic parameters, clinical features and quality of life, after the first injection of Botox until the 9 months of follow-up. Even after the second injection, with a follow-up of 3 months, we obtained results comparable with the first injection. Side-effects include an increase in post-void residual volume, dysuria and urinary infections. Conclusions We considered the dose of 100U of Botox, for treatment of IDO, as an efficacious and safe solution compared to other therapeutic options, without serious and lasting adverse effects for women, even after a second injection.

An increase in the serum concentration of pancreatic enzymes (amylase and lipase) was reported in a proportion of imatinib-resistant and/or intolerant Philadelphia-positive chronic myeloid leukemia patients treated with nilotinib. Acute pancreatitis was very rare, and the relevance of these laboratory alterations remains unknown. We report on 8 chronic myeloid leukemia patients who developed serum lipase/amylase elevation during treatment with nilotinib. After a median follow-up of 26 months, none of these patients developed an acute pancreatitis or clinical signs of pancreatic disease. Pancreatic hyperenzymemia never led to permanent drug discontinuation and required nilotinib temporary interruption in one case only. The median cumulative duration of dose interruptions and response to treatment were comparable in patients with or without pancreatic enzyme elevation. The mechanisms of action of nilotinib on pancreatic enzymes deserves to be investigated: however, in our experience, the relevance of pancreatic hyperenzymemia was clinically very limited.

Objective Urinary tract infections (UTIs) are common in the female population and, over a lifetime, about half of women have at least one episode of UTI requiring antibiotic therapy. The aim of the current study was to compare two different strategies for preventing recurrent bacterial cystitis: intravesical instillation of hyaluronic acid (HA) plus chondroitin sulfate (CS), and antibiotic prophylaxis with sulfamethoxazole plus trimethoprim. Materials and methods This was a retrospective review of two different cohorts of women affected by recurrent bacterial cystitis. Cases (experimental group) were women who received intravesical instillations of a sterile solution of high concentration of HA + CS in 50 mL water with calcium chloride every week during the 1 st month and then once monthly for 4 months. The control group included women who received traditional therapy for recurrent cystitis based on daily antibiotic prophylaxis using sulfamethoxazole 200 mg plus trimethoprim 40 mg for 6 weeks. Results Ninety-eight and 76 patients were treated with experimental and control treatments, respectively. At 12 months after treatment, 69 and 109 UTIs were detected in the experimental and control groups, respectively. The proportion of patients free from UTIs was significantly higher in the experimental than in the control group (36.7% vs. 21.0%; p = 0.03). Experimental treatment was well tolerated and none of the patients stopped it. Conclusion The intravesical instillation of HA + CS is more effective than long-term antibiotic prophylaxis for preventing recurrent bacterial cystitis.

The decrease of Chibby1 (CBY1) contributes to β catenin constitutive activation associated with the presence of the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML). This is mediated by transcriptional events and driven by DNA hyper-methylation at promoter-associated CpG islands of the CBY1-encoding gene C22orf2. Moreover, CBY1 transcriptional induction proceeding from promoter de-methylation is a component of BCR-ABL1+ cell response to Imatinib (IM). Our study showed that DNA methyltransferase 1 (DNMT1) has a central role in the hyper-methylation at the C22orf2 promoter. Further investigation in leukemic hematopoietic progenitors from IM-responsive and IM-resistant CML patients at diagnosis failed to demonstrate any correlation between DNMT1-driven hyper-methylation of the C22orf2 promoter and response to IM. Notably, the response to IM was neither predicted by DNMT1-driven hyper-methylation of BCL2-like11 at diagnosis. In conclusion, the hypermethylation of C22orf2 and BCL2-like11 promoters proceeding from DNMT1 is a crucial component of their reduced expression, but it is not directly involved in CML resistance to IM. It might rather contribute to the disease evolution towards a drug-resistant phenotype in more advanced phases or blast crisis.

The median age at diagnosis of chronic myeloid leukemia (CML) is between 60 and 65 years in most epidemiologic registries. Rather than age per se, a comprehensive evaluation of comorbidities may describe more properly the general clinical status of a patient. Tyrosine-kinase inhibitors (TKIs) have a different tolerability profile, and some adverse events (AEs) are peculiar of each drug, in particular, in presence of predisposing factors (comorbidities, concomitant medications). This article will review the impact of comorbidities in the safety and outcome of CML patients treated with TKIs. We will explore how the comorbidity status may be considered, together with CML-related factors, in the selection of the TKI in order to optimize treatment.

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.

We report a study of an alternative treatment schedule of imatinib (IM) in chronic myeloid leukemia (CML). Seventy-six Philadelphia-positive (Ph+), BCR-ABL-positive patients aged 65 years or older who had been treated with IM for more than 2 years and who were in stable complete cytogenetic response (CCgR) and major molecular response (MMR) were enrolled in a single-arm study to test the effects of a policy of intermittent IM (INTERIM) therapy for 1 month on and 1 month off. With a minimum follow-up of 4 years, 13 patients (17%) lost CCgR and MMR and 14 (18%) lost MMR only. All these patients resumed continuous IM and all but one (lost to follow-up) regained CCgR and MMR. No patients progressed to accelerated or blastic phase or developed clonal chromosomal abnormalities in Ph+ cells or BCR-ABL mutations. In elderly Ph+ CML patients carefully selected for a stable CCgR (lasting >2 years), the policy of INTERIM treatment affected the markers of residual disease, but not the clinical outcomes (overall and progression-free survival). This trial was registered at www.clinicaltrials.gov as NCT 00858806.

We investigated whether low-level Bcr–Abl kinase domain mutations can be detected in patients who have stable responses to first-line nilotinib–like it is known to happen in patients receiving imatinib. We screened for mutations twelve such patients by cloning and sequencing. Only one case was found to harbor mutations at low levels (including a T315I). However, major molecular response (MMR) was maintained and it even improved to complete molecular response. Our results suggest that a) Bcr–Abl mutations, even at low level, seem to be very rare in patients in MMR on first-line nilotinib; b) low-level mutations do not always predict for subsequent relapse.

Abstract 112 BCR-ABL kinase domain (KD) mutation analysis may be an useful tool for physicians and is being performed in a growing number of laboratories. Recommendations aimed to rationalize the use of mutation testing in chronic myeloid leukemia (CML) have recently (Blood 2011) been compiled by a panel of experts appointed by European LeukemiaNet (ELN) – including specific recommendations as to when mutation analysis should be performed. They came from the expert opinion of the panel members whenever published data were insufficient or contradictory. In order to provide further data to validate or refine these recommendations, we have analyzed the GIMEMA CML WP database recording the results of mutation analyses performed in CML pts (n=1301) receiving imatinib and/or 2nd generation TKIs between January 2004 and July 2011. At dagnosis, mutation analysis was recognized to be useful in the few pts who present in accelerated phase or blast crisis (BC), while it was not recommended in chronic phase (CP) pts. Interrogating our database, we could retrieve 58 mutation analyses in newly diagnosed pts in CP and 12 in newly diagnosed pts in BC. Imatinib-resistant mutations were detected in 0 and 2 pts, respectively. In pts receiving 1st-line imatinib, mutation analysis was recommended both in case of failure and in case of suboptimal response. We have analyzed 399 chronic phase (CP) CML pts receiving first-line imatinib because they were found to meet one of the criteria for failure or suboptimal response. Overall, 45/166 (27.1%) failures were found to be positive for one or more BCR-ABL KD mutations. In particular, mutations were detected in 3/16 (18.8%) pts with less than CHR at 3 months, 1/9 (11.1%) pts with no CyR at 6 months, 4/24 (16.7%) pts with less than PCyR at 12 months, 6/36 (16.7%) pts with less than CCyR at 18 months, 15/49 (30.6%) pts who lost CCyR and 16/32 (50%) pts who lost CHR. More interestingly, only 11/233 (4.7%) suboptimal responders we analyzed were positive for mutations. Among ‘cytogenetic' suboptimal responders, mutations were detected in 1/15 (6.7%) pts with no CyR at 3 months, 1/20 (5.0%) pts with less than PCyR at 6 months, 5/51 (8.2%) pts with less than CCyR at 12 months. Among ‘molecular' suboptimal responders, mutations were detected in 0/52 pts with less than MMR (but having achieved CCyR) at 18 months and in 4/95 (4.2%) pts who lost MMR (but not CCyR). Which rise in Bcr-Abl transcript level should trigger a mutation analysis was the most difficult issue to provide recommendations upon, given the lack of convincing and reproducible data in the literature. It was finally agreed to recommend mutation analysis only in case of MMR loss. In 159 of the CP pts we have analyzed, mutation analysis was specifically requested because of a transcript increase at a single RQ-PCR assessment: 29 pts had less than 1-log increase and 41 pts had a 1-log increase or more – but with no loss of MMR. None of these pts was found to have mutations. Another 36 pts had less than 1-log increase and 53 had a 1-log increase or more, leading to loss of MMR. Mutations were detected in 1 (2.8%) and 3 (5.7%) pts, respectively. In pts receiving dasatinib or nilotinib as 2nd-line agents, mutation analysis was recommended at baseline and then in case of failure according to the provisional definitions proposed by Baccarani et al (J Clin Oncol 2009). Nineteen among the pts we analyzed met these criteria; overall, mutations were detected in 11 (57.8%), including 5/7 pts with no CyR at 3 months, 6/9 pts with minimal CyR at 6 months, 1/4 pts with less than PCyR at 12 months. In addition, newly acquired mutations were detected in 93/131 (71%) pts who lost a previously achieved HR or CyR. We also tested 19 pts who met the provisional definitions for suboptimal response to dasatinib or nilotinib 2nd-line. Mutations were detected in 4/19 pts (21%), including 2/5 pts with minor CyR at 3 months, 1/7 pts with PCyR at 6 months, 1/7 pts with less than MMR at 12 months. Our data indicate that: a) pts harbouring mutations can more frequently be found among cytogenetic suboptimal responders than among molecular suboptimal responders; b) any Bcr-Abl transcript increase that is not associated with MMR loss shouldn't indeed trigger a mutation analysis; c) although definitions of response to dasatinib or nilotinib 2nd-line are still provisional and might soon be refined, not only failures but also suboptimal responses are frequently associated with mutations. Supported by AIL, AIRC, PRIN and FIRB. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Martinelli:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

Imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, some patients may experience resistance or intolerance to imatinib over time. For these patients, two excellent therapeutic options are available. Dasatinib, approved in 2006 for the treatment of patients with CML in all phases who experience imatinib resistance or intolerance, has displayed significant efficacy, with a 2-year follow-up showing durable hematologic and cytogenetic responses, as well as prolonged progression-free and overall survival. Nilotinib was approved in 2007 for the treatment of patients with CML in chronic phase or CML in accelerated phase, resistant or intolerant to prior therapy including imatinib, based on strong efficacy as well as a favorable safety profile. Several factors, including mutation status, patient history, and existing comorbidities can impact the decision to use dasatinib or nilotinib, or pursue other options such as allogeneic stem cell transplant. The purpose of this review article is to shed light on a further consideration when deciding which agent to use, based on the efficacy and safety of dasatinib and nilotinib. This consideration involves the differences in both the imatinib-resistant and -intolerant patients enrolled in the pivotal studies for each agent, as well as other trial criteria.

Imatinib is the standard front-line therapy of chronic myeloid leukaemia (CML). The evaluation of the response is based on blood counts and differential (haematologic response, HR), on the examination of marrow cell metaphases (cytogenetic response, CgR) and on a quantitative assessment of BCR-ABL transcripts level (molecular response, MolR). An optimal response to imatinib is defined by complete HR and at least minimal CgR (Ph +95%) at 3 months, at least partial CgR (Ph +35%) at 6 months, complete CgR at 12 months and major MolR (BCR-ABL: ABLor = 0.1%) at 18 months. Failure is defined by incomplete HR at 3 months, no CgR (Ph +95%) at 6 months, less than partial CgR (Ph +35%) at 12 months, less than complete CgR at 18 months and loss of a complete HR or a complete CgR. In any other situation, the response is defined suboptimal. Treatment recommendations are to continue on imatinib in case of optimal response and to move to second-generation tyrosine kinase inhibitors (TKIs) and/or to allogeneic haematopoietic stem cell transplantation in case of failure. In case of suboptimal response, treatment may be continued with imatinib, at the same dose or a higher dose, but some patients may become eligible for second-generation TKIs. A provisional definition of the response to second-generation TKIs second line is provided.

Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

We assessed the capacity of positively selected autologous CD133(+) hematopoietic stem cells (HSCs) to reconstitute lymphomyelopoiesis in chronic lymphocytic leukemia (CLL) patients receiving myeloablative chemotherapy. Ten resistant/relapsed CLL patients underwent HSC mobilization with chemotherapy and granulocyte-colony stimulating factor (G-CSF). Positive selection of circulating CD133(+) HSCs was performed by immunomagnetic technique. Highly purified HSCs were reinfused after busulphan/melphalan myeloablative treatment. A median number of 4.2 x 10(6) CD34(+) cells/kg and of 3.14 x 10(6) CD133(+) cells/kg were collected. Immunomagnetic selection resulted in the reinfusion of a median number of 2.45 x 10(6) CD133(+) cells/kg (median purity: 94.8%; median recovery: 84%) and 2.4 x 10(6) CD34(+) cells/kg (median purity: 93%; median recovery: 71%). HSC selection resulted in a median T cell and CD19(+)/CD5(+) cell depletion of 3.85 log and 2.8 log, respectively. At the molecular level, however, 7 of 8 valuable purified HSC fractions were contaminated by leukemic cells. All CLL patients showed rapid and sustained myeloid engraftment after reinfusion of purified CD133(+) cells. Immunologic reconstitution was comparable to that routinely observed in patients reinfused with unmanipulated leukapheresis products and no late infectious complications were observed. With a median follow-up of 28 months for transplanted patients, 5 patients are in clinical complete remission, 3 are in partial remission, and 1 is in progression. In conclusion, the reinfusion of highly purified CD133(+) HSCs allowed the rapid and sustained recovery of hematopoiesis after myeloablative treatment in resistant/relapsed CLL patients. However, the purging potential of positive selection of CD133(+) cells is not adequate to achieve tumor-free autografts.

Summary Fifteen multiple myeloma (MM) patients who had failed maintenance therapy after tandem autologous stem cell transplantation underwent anti-idiotype (Id) vaccination with dendritic cells (DCs). CD14+-derived DCs were loaded with the autologous Id as whole protein (=6) or Id-derived class I-restricted peptides (=9) and keyhole limpet hemocyanin (KLH). Vaccination consisted of three subcutaneous (sc) and two intravenous injections of increasing DC doses at 2 weeks interval. DC therapy was well tolerated. Most patients developed both humoral and T-cell responses to KLH, suggesting immunocompetence. Eight of 15 patients developed an Id-specific T-cell proliferative response, 8/15 increased interferon-γ-secreting T cells and 4/15 showed an Id-positive delayed-type hypersensitivity test. Anti-Id cytotoxic T-lymphocyte precursors increased after DC vaccination in 2/2 evaluable patients. A more robust T-cell response was observed after sc DC injections and increased Id-specific T-cell proliferation was found up to 1 year after vaccination. VDJ-derived peptides were as effective as the whole protein in stimulating T-cell responses. Clinically, 7/15 patients have stable disease after a median follow-up of 26 months, one patient achieved durable partial remission after 40 months, and seven patients progressed. In conclusion, sc injections of cryopreserved Id-pulsed DCs were safe and, in contrast with intravenous administrations, induced anti-MM T-cell responses.

The median age at diagnosis of Chronic Myeloid Leukemia (CML) reported in clinical trials is generally of 50-55 years; it is higher (more than 60 years) in epidemiologic registries and in observational studies [1]. Therefore, a significant proportion of CML patients are “elderly”, according to the most widely accepted definition of “old person” (age > 65 years). Prior to imatinib (IM) introduction, an orally taken tyrosine kinase inhibitor, older age was associated to a worse outcome (lower response rates and lower long-term survival), regardless of the treatment used (busulfan, hydroxyurea, interferon alpha), basically, for unknown reasons. It can be speculated that, with interferon, the reason of an inferior outcome of elderly patients lied in a poorer compliance; however, this worsen prognosis of elderly CML patients have been described even employing well tolerated drugs (like hydroxyurea). Age was included in the 2 most used risk scores for CML: Sokal score (1984), referring to patients treated with conventional chemotherapy, and EURO score (1998), for patients treated with interferon [2, 3]. The introduction of IM has revolutionized the therapy of CML patients, significantly improving survival [4]. However, in the early days of IM, many physicians felt that the prognosis of an old CML patient, particularly those older than 75-80 yrs, could not be positively influenced by IM and a not negligible part of elderly patients are still not allocated to IM (or enrolled in clinical trials) for different reasons (co-morbidities, toxicity, long term outcome, socio-economic factors) [5]. The importance of age in the IM era has been investigated in different studies, leading to a re-evaluation of its prognostic significance. A large study in late chronic phase patients treated with IM after interferon failure, conducted by GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) CML Working-Party (WP), showed lower hematologic and cytogenetic response rates in older patients (> 65 years old), but similar overall survival with a 36 months median follow-up [6]. In another large but heterogeneous (early chronic phase, late chronic phase, and advanced phases) series of patients treated with IM at the MDACC, older age appeared to be not associated with a worse outcome; however, the cut-off to define older patients was 60 years, elderly early chronic phase patients were few (49) and no long term data were available [7]. The first study to evaluate the effect of age in large series of front-line IM treated patients and with a long follow-up was recently published by the GIMEMA CML WP [8]. Five hundred and fifty nine patients, of whom 115 ≥ 65 years old, were enrolled in 3 prospective, multicenter, trials. After a median follow-up of 60 months, hematologic, cytogenetic and molecular response rates were identical in older and younger patients while survival was significantly inferior in older patients. This lower survival was due to a higher number of deaths occurred in chronic phase, reasonably unrelated to IM therapy or toxicity, and not to a higher progression rate in older patients. Recently a new prognostic score was elaborated based on 2060 patients enrolled in studies of first-line IM-based regimes and collected by the European Leukemia Net / EUTOS Registry [9]. Of the all variables analyzed, only spleen size and basophils percentage, best discriminated between high-risk and low-risk patients; age had no more a prognostic impact, a further confirmation that IM has cancelled the differences in prognosis between younger and older patients. No doubt, the overall toxicity and the need for dose optimization are higher for IM with respect to conservative and palliative treatments, like hydroxyurea, particularly when older patients are treated; however, in the hands of expert physicians IM is generally manageable. Moreover, as the lower survival of elderly patients is mainly determined by age-related factors and co-morbidities, rather than by the progression of CML, a geriatric assessment (frailty status) may be helpful in the decision to allocate selected patients to IM therapy [10]. In conclusion, age had relevance when therapeutic strategies, like IM, were not available. However, today, older age per se must not be a limitation for treating patients with IM and the enrollment of these patients in clinical trials should be encouraged. Second generation tyrosine kinase inhibitors (dasatinib and nilotinib) as front-line therapy showed higher response rates and lower toxicities compared to IM [11, 12]; probably these results will be confirmed also in elderly patients. Importantly, extra-hematologic toxicities are distinct between IM, dasatinib and nilotinib, allowing the selection of the more appropriate drug in relation to the presence of co-morbidities. Although data on dasatinib and nilotinib in elderly patients are still few and the follow-up is still short, these second generation tyrosine kinase inhibitors will probably further improve the outcome of CML elderly patients.

Abstract 2720 Treatment strategies based on second generation tyrosine kinase inhibitors such as Nilotinib, have improved overall chronic myeloid leukemia (CML) treatment results, with a rapid and major molecular remission (MCR), at 12 months rate ranging 75%. Therefore, the detection of residual leukemic cells by measuring BCR-ABL1 transcript level is absolutely required to monitor minimal residual disease. To investigate the molecular therapeutic efficacy of nilotinib 400 mg BID in previously untreated, ECP, Philadelphia-positive CML patients, the Italian GIMEMA CML Working Party is conducting an multicenter phase II study (ClinicalTrials.gov NCT00481052). The primary endpoint is the CCgR rate at 1 year. The kinetic of molecular response is being studied by Q-PCR at baseline and after 1, 2, 3, 6, 9, 12, 18 and 24 months from treatment start by a TaqMan absolute quantitative PCR (Applied Biosystems 7900HT Fast Real-Time PCR System) and by the 12.765 Digital array (Fluidigm) which is is a nanofluidic biochip consisting in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Fluidigm analysis was done in parallel on same samples (at diagnosis,1, 2, 3, 6, 9, 12, and were possible at 18 and 24 months). Patient with longest follow up was 727 days. A Major Molecular Response, defined as a BCR-ABL:ABL ratio The molecular results achieved in our study strongly support the notion that in early chronic phase CML patients molecular responses to nilotinib are substantially faster and “deeper” than those to IM. More rapid reduction of residual disease might help to reduce failures and to improve the late outcome of therapy. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: No relevant conflicts of interest to declare.

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

BACKGROUND: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. METHODS: a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. RESULTS: DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. CONCLUSIONS: DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.