Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia

We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future

Outcome of Patients with CML Treated with Dasatinib or Nilotinib after Failure of Second Prior TKIs.

Background The TKIs Nilotinib and Dasatinib offer additional therapeutic options for patients with CML who are resistant or intolerant to Imatinib. These agents, active against the majority of Imatinib resistant BCR-ABL mutated clones, have a different pattern of kinase target selectivity, pharmacokinetics parameters, cell uptake, efflux properties and adverse events profiles. Preliminary results suggest that some patients may respond to a second TKI used as third line therapy ,but little is known about the long term benefit of such an approach.Aim of this collaborative Italian study was to verify the response (rate and duration) and the clinical outcome in patients with CML treated with a third TKI after sequential failure of the previous ones. Methods. We evaluated 66 patients with CML, resistant/intolerant to Imatinib and treated with Dasatinib or Nilotinib, then switched to a third- line TKI after treatment failure. Of these, 29 patients were treated with dasatinib after imatinib/nilotinib failure and 37 with nilotinib after imatinib/dasatinib failure. Patients were monitored with complete blood counts, cytogenetic analysis, bone marrow aspiration RT-PCR and mutational analysis. Results. A total of 66 patients (median age 63 years, range, 33-85 years) were treated with sequential TKIs; 40 (61%) patients had received interferon-a before starting Imatinib; 26 (39%) patients received imatinib as first line therapy. The median time on imatinib therapy was 47.5 months (range 4-101 months). At the start of nilotinib as second line, 27/29 (93%) patients were in CP, 1 (3.5%) in AP, and 1 (3.5%) in BP. 9 patients (31%) had developed mutations before starting treatment. The median time on second line TKI was 8 months (range 2-36 months). In the resistant patients 4 new mutations were identified (F359V in two patients, T315I, Y253H+F359V). At the start of dasatinib as second line, 33/37 (89.2%) patients were in CP, 4 (10.8%) in AP. 7 patients (18.9%) had developed mutations before starting treatment. The median time on second line TKI was 14 months (range 4-59 months).In the resistant patients 5 new mutations were identified (F137L in three pts, M318T, M244V+F317L). At the start of the third TKI, 60/66 (90.9%) patients were in CP, 5 (7.6%) in AP, and 1 (1.5%) in BP. Of these, 7 patients (18.9%) on dasatinib and 7 (24.1%) on nilotinib had mutations before starting treatment. The best response to the third line treatment with TKI was 10 (15.2%) MMR, 10 (15.2%) CCyR, 8 PcyR (12.1%), 5 (7.5%) mCyR, 24 (36.4%) CHR and 9 (13.6%) No Response (NR). In the dasatinib group, 9 (31%) patients discontinued treatment because of toxicity versus 17 (45.9%) patients in the nilotinib group.Two new mutations (F317L, E255V) emerged with dasatinib as third line therapy.After a median follow up of 13 months (range 2-37 months) 50 patients (48 CP, 2 AP) are continuing therapy ( 33 on nilotinib, 17 on dasatinib).Since the start of the third TKI, 61 patients (92.4%) are still alive for a median overall survival of 110 months( range 15-300) (52 CP, 7 AP, 2 NA); the 5 deaths (7.6%) were caused by disease progression and spread of the gene mutation T315I. Discussion.In our study, about one third of patients derived benefit from the use of three sequential TKIs; patients with better, longer response (28.7%) to third TKI were the same patients with a better response to the Imatinib and 2TKIs therapy. All these patients had taken interferon therapy before the Imatinib. In this subset of patients (good responders: CCyR and MMR) 5 patients developed mutations that were sensitive to the sequential treatment.The lack of a durable cytogenetic remission could be explained by the emergence of new kinase domain mutations as patients are exposed to sequential TKI; a change of therapy resulted in an adequate response. In our series, patients with poor prognosis showed mutations not sensitive to the TKIs treatment. Conclusions. Although allogeneic SCT is the treatment of choice in all patients failing 2 TKIs who are suitable candidates for this approach, alternative strategies are required for ineligible patients. The use of a third TKI after failure of two previous TKIs induces response in some patients. Longer follow up of a larger series of patients is needed to determine the long term impact of the response

Results of a Multicenter, Controlled, Randomized Clinical Trial Evaluating the Combination of Piperacillin/Tazobactam and Tigecycline in High-Risk Hematologic Patients With Cancer With Febrile Neutropenia

Purpose Empiric antibiotic monotherapy is considered the standard of treatment for febrile neutropenic patients with cancer, but this approach may be inadequate because of the increasing prevalence of infections caused by multidrug resistant (MDR) bacteria. Patients and Methods In this multicenter, open-label, randomized, superiority trial, adult, febrile, high-risk neutropenic patients (FhrNPs) with hematologic malignancies were randomly assigned to receive piperacillin/tazobactam (4.5 g intravenously every 8 hours) with or without tigecycline (50 mg intravenously every 12 hours; loading dose 100 mg). The primary end point was resolution of febrile episode without modifications of the initial allocated treatment. Results Three hundred ninety FhrNPs were enrolled (combination/monotherapy, 187/203) and were included in the intention-to-treat analysis (ITTA). The ITTA revealed a successful outcome in 67.9% v 44.3% of patients who had received combination therapy and monotherapy, respectively (127/187 v 90/203; absolute difference in risk (adr), 23.6%; 95% CI, 14% to 33%; P < .001). The combination regimen proved better than monotherapy in bacteremias (adr, 32.8%; 95% CI, 19% to 46%; P < .001) and in clinically documented infections (adr, 36%; 95% CI, 9% to 64%; P < .01). Mortality and number of adverse effects were limited and similar in the two groups. Conclusion The combination of piperacillin/tazobactam and tigecycline is safe, well tolerated, and more effective than piperacillin/tazobactam alone in febrile, high-risk, neutropenic hematologic patients with cancer. In epidemiologic settings characterized by a high prevalence of infections because of MDR microorganisms, this combination could be considered as one of the first-line empiric antibiotic therapies