Philadelphia-positive patients who already harbor imatinib-resistant Bcr-Abl kinase domain mutations have a higher likelihood of developing additional mutations associated with resistance to second- or third-line tyrosine kinase inhibitors

Abstract 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)

The efficacy of two methods of exercise training in moderate/severe COPD

none5The efficacy of two methods of exercise training in moderate/severe COPD. Introduction: Exercise training (ET) is the key component of respiratory rehabilitation. In cost-benefit terms the ET programs are advantageous if carried out at home. This can be difficult for patients, especially in terms of understanding exercise intensity. Aim: To evaluate two methods of home ET (walking). Methods: 33 COPD. Group A: 19 (7F,12M) participated to the ET program (age 74 ±1,3; FEV1% 46,4±3,6); Group B: 14 (2F,12M) did not participate (age 71 ±1,2; FEV1% 55,4±3,5). After the initial evaluation and the assessment of optimal speed walking (T0), patients received a home ET program and were studied after 5 months (T5), by means of spirometry, Saint George Questionnaire (SGQR), treadmill walking test and Armband to monitor physical activity. Patients were further divided in 2 groups: A1) speed walking marked by a metronome; A2) covering a known distance in a fixed time. Results: Group A (Table1), (Table 2). SGQR score FEV1 (L/sec) Daily METs Physical activity > 3 METs (min) Distance (m) Speed (Km/h) T0 35,2±4 1,06±0,08 1,26±0,04 22,2±5,4 449±50 1,7±0,12 T5 27,4±2,6* 1,13±0,07* 1,38±0,06*** 44,4±10,2** 891±91*** 2,7±0,2*** METs Metabolic Equivalent: 3.5 ml O2/Kg/min or 1 Kcal/kg/h Δ % Distance Δ % Speed A1 15,1±6* 14,5±5,7* A2 1,9±6,7 1,8±5,5 Group B: no significant change. Conclusions: As expected, the ET improved exercise capacity and QoL but patients using the metronome improved significantly more than others. ET performed at a metronome-marked speed is more effective. Statistical Analysis by T-Test: significance *=p<0,05 **=p<0,01 ***=p<0,001noneL. Pomidori;G. Mandolesi;M.C. Gnani;A. Gennari; A. CogoPomidori, Luca; G., Mandolesi; Gnani, Maria Cristina; Gennari, Alessandra; Cogo, Annaluis

Gut Microbiota Profiling of Pediatric Nonalcoholic Fatty Liver Disease and Obese Patients Unveiled by an Integrated Meta-omics-Based Approach

There is evidence that non-alcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in paediatric NAFLD patients using targeted-metagenomics (MG) and metabolomics (MB). Stools were collected from 61 consecutive patients diagnosed with NAFL, NASH, or obesity and 54 healthy subjects (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds (VOCs) determined by solid-phase micro-extraction GC-MS. The α-diversity was highest in CTRLs followed by obese, NASH, NAFL patients and β-diversity distinguished between patients and CTRLs, but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences amongst NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing MG and MB data dimensionality, multivariate analyses indicated Oscillospira decrease in NAFL and NASH groups, and Ruminococcus, Blautia, and Dorea increase in NASH patients compared to CTRLs. Of the 292 VOCs, 26 were up- and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea, Ruminococcus and higher levels of 2-butanone, 4-methyl-2-pentanone compared to CTRLs

Low level mutations in the Bcr-Abl kinase domain may already be detected at diagnosis both in patients with Philadelphia-positive acute lymphoblastic leukemia and in patients with chronic phase chronic myeloid leukemia

Mutations in the Bcr-Abl kinase domain (KD) are often detected at the time of resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). It is still unclear whether and in how many pts low level mutations may already be detectable at the time of diagnosis. We therefore analyzed cDNA samples from 22 newly diagnosed pts with Ph+ ALL (n=13) or chronic phase (CP)-CML (n=9) who subsequently received TKI therapy (imatinib, dasatinib or nilotinib). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 independent clones for each pt. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. Each mutation was present in two to five independent clones. A total of 105 mutations (including 35 silent, 5 nonsense, and 65 missense mutations) were observed. The vast majority (98/105, 93%) of them have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 93/105 (89%) mutations were transitions: G>A (n=28), A>G (n=23), C>T (n=22), T>C (n=20). Such a high prevalence of transitions (normally occurring 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is active in Ph+ cells. One of the nine CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with evidence of mutations, but only one with a mutation (T315I) that was already detectable at diagnosis. The remaining eleven pts are in persistent remission after a follow up ranging from 12 to 36 months, although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. Our observations suggest that: a) Bcr-Abl KD mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent or not conferring any growth advantage under the selective pressure of TKIs; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all CML and Ph+ ALL pts before the start of TKI therapy

Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis

BACKGROUND: In patients with Philadelphia-positive acute lymphoblastic leukemia, resistance to treatment with tyrosine kinase inhibitors is frequent and most often associated with the development of point mutations in the BCR-ABL kinase domain. We aimed to assess: (i) in how many patients BCR-ABL kinase domain mutations are already detectable at relatively low levels at the time of diagnosis, and (ii) whether mutation detection correlates with subsequent response to therapy. DESIGN AND METHODS: We retrospectively analyzed samples collected at diagnosis from 15 patients with Philadelphia-positive acute lymphoblastic leukemia who subsequently received tyrosine kinase inhibitor therapy (dasatinib) by cloning the BCR-ABL kinase domain in a bacterial vector and sequencing 200 independent clones per sample. RESULTS: Mutations at relatively low levels (2-4 clones out of 200) could be detected in all patients--eight who relapsed and seven who achieved persistent remission. Each patient had evidence of two to eight different mutations, the majority of which have never been reported in association with resistance to tyrosine kinase inhibitors. In two patients out of six who relapsed because of a mutation, the mutation (a T315I) was already detectable in a few clones at the time of diagnosis. On the other hand, a patient who was found to harbor an F317L mutation is in persistent remission on dasatinib. CONCLUSIONS: Our results suggest that the BCR-ABL kinase domain is prone to randomly accumulate point mutations in Philadelphia-positive acute lymphoblastic leukemia, although the presence of these mutations in a relatively small leukemic subclone does not always preclude a primary response to tyrosine kinase inhibitors

Efficacy and clinical outcome of Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) patients treated with second generation tyrosine kinase inhibitors (TKIs): The Bologna experience

Background. Approximately 30% of adult ALL patients are characterized by the presence of the Philadelphia (Ph) chromosome, which derives from a reciprocal translocation t(9;22)(q34;q11) and results in a chimeric BCR-ABL oncogene. The prognosis of this subset of patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs