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Purpose: Most patients with chronic-phase chronic myeloid leukemia (CML) who receive imatinib achieve a complete cytogenetic remission (CCgR) and low levels of BCR-ABL transcripts. CCgR is durable in the majority of patients but relapse occurs in a subset. Experimental Design: To determine the potential of quantitative reverse transcription-PCR of BCR-ABL to predict cytogenetic relapse, we serially monitored residual disease in 97 CML patients with an imatinib-induced CCgR. Patients with late chronic phase CML after IFN-α failure were treated with imatinib (400 mg daily). Results: During the imatinib median follow-up time of 36 months (range, 12-54 months), disease monitoring occurred by cytogenetics and quantitative PCR. Twenty percent of patients experienced cytogenetic relapse at a median of 18 months after CCgR and a median of 24 months after starting imatinib. None of the possible prognostic factors studied in univariate and multivariate analyses seemed to predict for loss of cytogenetic response but the reduction of BCR-ABL transcript levels at the time of CCgR is an important prognostic factor. Conclusions: In our study, we showed not only that achieving a major molecular remission at 12 months is predictive of a durable cytogenetic remission but also that patients who achieved a major molecular remission (expressed both as the BCR-ABL/β2 microglobulin ratio %

Dasatinib is a potent BCR-ABL inhibitor effective in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL) resistant/intolerant to imatinib. In the GIMEMA LAL1205 protocol, patients with newly diagnosed Ph+ ALL older than 18 years (with no upper age limit) received dasatinib induction therapy for 84 days combined with steroids for the first 32 days and intrathecal chemotherapy. Postremission therapy was free. Fifty-three patients were evaluable (median age, 53.6 years). All patients achieved a complete hematologic remission (CHR), 49 (92.5%) at day 22. At this time point, 10 patients achieved a BCR-ABL reduction to < 10−3. At 20 months, the overall survival was 69.2% and disease-free survival was 51.1%. A significant difference in DFS was observed between patients who showed at day 22 a decrease in BCR-ABL levels to < 10−3 compared with patients who never reached these levels during induction. In multivariate analysis, BCR-ABL levels of < 10−3 at day 85 correlated with disease-free survival. No deaths or relapses occurred during induction. Twenty-three patients relapsed after completing induction. A T315I mutation was detected in 12 of 17 relapsed cases. Treatment was well tolerated; only 4 patients discontinued therapy during the last phase of the induction when already in CHR. In adult Ph+ ALL, induction treatment with dasatinib plus steroids is associated with a CHR in virtually all patients, irrespective of age, good compliance, no deaths, and a very rapid debulking of the neoplastic clone. This trial was registered at [www.clinicaltrials.gov][1] as #[NCT00391989][2]. [1]: http://www.clinicaltrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00391989&atom=%2Fbloodjournal%2F118%2F25%2F6521.atom

Background: Although recent high-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has contributed to the identification of many novel somatic genetic alterations, a deep molecular characterization of adult ALL is still challenging, especially for those cases lacking recurrent fusion genes. Aims: In order to better molecularly dissect this ALL subgroup, we performed an integrative molecular approach including gene-candidate high-resolution screening and genome-wide profiling analyses. Methods: We retrospectively analyzed 28 newly diagnosed BCR-ABL1- negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%). Copy number alterations (CNA) of know leukemiarelated genes, such as IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA have been assessed by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, sequence mutations have been investigated in TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by nextgeneration deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5- JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix), gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) and whole exome sequencing (Illumina) were performed to more fully assess genomic complexity. Results: Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed known leukemia genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of JAK2 and CRLF2 in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p

Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and no serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and no serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level. ispartof: BMC Genomics vol:15 issue:1 pages:487-487 ispartof: location:England status: published

Hypomethylating agents, such as 5-Azacytidine (AZA), significantly modified the therapeutic approach to MDS, primarily in older patients with higher risk disease for whom intensive chemotherapy and allogeneic stem cell transplantation are not an option. In low-risk MDS, 5- AZA aims to reduce transfusion dependency, improve quality of life and hopefully the survival, but it is still unclear if this therapeutic approach would be cost-effective. At a molecular level, the mechanisms underlying the effect of epigenetic therapy are not completely understood, although it is well known that DNA methyltransferase inhibitors can induce the expression of Phosphoinositide-Phospholipase C (PIPLC) beta1 in high-risk MDS. Here, we prospectively investigated the efficacy and safety of AZA in low-risk MDS patients. AZA was administered at a lower intensity schedule, that is 75 mg/sqm/day subcutaneous for 5 days every 28, for a total of 8 cycles, and response was assessed at the 4th and 8th cycle of AZA. Moreover, PI-PLCbeta1 promoter methylation and gene expression levels were quantified before and after each cycle of 5-AZA. The study included 32 patients, and 26 cases completed 8 cycles of AZA. ORR was 47% (15/32) on intention to treat and 58% (15/26) for patients completing the treatment program. In this latter group, 5 (19%) cases achieved CR and 10 (38%) had HI, according to the IWG criteria. Interestingly, three patients have maintained their HI after 37, 34 and 33 months without other treatments. At a molecular level, although baseline PI-PLCbeta1 levels were not correlated to clinical response, 5d-AZA induced a statistically significant decrease in PI-PLCbeta1 promoter methylation in 14/15 responders, which corresponded to a significant increase in PI-PLCbeta1 mRNA. In 9/14 (64%) responsive patients, the first molecular increase in PI-PLCbeta1 level was observed between the 3rd and 4th cycle, therefore anticipating the clinical evaluation. In addition, 8 cases showed a loss of the response after the end of therapy (8th cycle) and these cases displayed a significant reduction of PI-PLCbeta1 levels, below the pre-treatment values, already before the clinical loss of the response. Taken together, our results show that 5d-AZA is safe and effective in a proportion of low risk MDS patients. PI-PLCbeta1 gene expression is a reliable and dynamic marker of response that can be useful to optimize AZA therapy.

Introduction. T-ALL is an aggressive malignancy and despite improvements in survival rates prognosis is still poor. A common aberrantly expressed pathway related to T-ALL and converging on anti-apoptotic and pro-survival signals activation is PI3K/Akt and its inhibition is an attractive strategy to improve current treatments. NVP-BKM120 is a highly selective pan-class I PI3K inhibitor which has shown antitumor activity in preclinical and clinical studies in solid cancers. Here we investigated its efficacy in T-ALL. Methods. A panel of T-ALL cell lines with up-regulated PI3K/Akt signaling and primary T-ALL blasts were treated with increasing concentrations of NVP-BKM120 and analyzed at different time points. Results. MTT assays documented a strong reduction of viability in all cell lines tested with a median IC50 lower than 2μM after 48 hours treatment that correlate with apoptosis as documented by Annexin V/PI analysis and activatation of caspases. To asses selective inhibition of PI3K, levels of the pathway components p-Akt(ser473) and p-S6RP(Ser235/236) have been evaluated by western blotting showing a strong decrease in a dose and time dependent manner. Efficacy of NVP-BKM120 was also tested in an ex-vivo model of primary blasts from T-ALL patients with assessed constitutive activation of PI3K/Akt pathway and results were consistent with in vitro studies. Comparison between NVP-BKM120 and PI3K selective inhibitors p110 isoforms provided its stronger effect in terms of viability and pathway inhibition. The drug was also able to synergize with dexamethasone and vincristine both in vitro and ex-vivo. To note, NVP-BKM120 extents pro-apoptotic effects also in Jurkat cells co-cultured with MS-5 stromal cells which mimic bone marrow microenvironment suggesting a potential overcome of its protective effect toward leukemic cells in vivo. Finally cell cycle has been analyzed by flow cytometry revealing a strong accumulation in the G2/M phase while immunofluorescent analysis identified an increased number of mitotic cells with disorganized mitotic spindle compared to control, suggesting an impairment in G2/M regulation and in mechanisms involved in cell cycle progression. Conclusion: NVPBKM120 showed viability reduction and apoptosis induction in T-ALL cells as well as in primary T-ALL blasts due to PI3K pathway inhibition supporting its clinical evaluation in T-ALL.

For almost 10 years imatinib has been the therapeutic standard of chronic myeloid leukemia. The introduction of other tyrosine kinase inhibitors (TKIs) raised a debate on treatment optimization. The debate is still heated: some studies have protocol restrictions or limited follow-up; in other studies, some relevant data are missing. The aim of this report is to provide a comprehensive, long-term, intention-to-treat, analysis of 559 newly diagnosed, chronic-phase, patients treated frontline with imatinib. With a minimum follow-up of 66 months, 65% of patients were still on imatinib, 19% were on alternative treatment, 12% died and 4% were lost to follow-up. The prognostic value of BCR-ABL1 ratio at 3 months (⩽10% in 81% of patients) was confirmed. The prognostic value of complete cytogenetic response and major molecular response at 1 year was confirmed. The 6-year overall survival was 89%, but as 50% of deaths occurred in remission, the 6-year cumulative incidence of leukemia-related death was 5%. The long-term outcome of first-line imatinib was excellent, also because of second-line treatment with other TKIs, but all responses and outcomes were inferior in high-risk patients, suggesting that to optimize treatment results, a specific risk-adapted treatment is needed for such patients.

Background. The use of hypomethylating agents significantly modified the therapeutic approach to MDS patients, primarily in high-risk MDS patients. In low-risk MDSs the use of AZA hypomethylating agent is less understood. Epigenetic regulation of Phosphoinositide-Phospholipase C (PI-PLC) beta1 promoter and key molecules involved in the nuclear inositide signalling pathways seems to play an important role for the activity of AZA demethylating therapy. Aims. We prospectively evaluated the efficacy and safety of AZA low-dose in Low or Int-1 risk MDS patients who were symptomatic and/or unresponsive to previous treatments. In the same study we evaluated the molecular effects of AZA on PI-PLCbeta1 promoter methylation, in order to investigate a possibly correlation with the response to the drug. Methods. AZA was administered at a dose of 75 mg/mq/daily s.c for 5 consecutive days every 28 days for a total of 8 cycles. Final response was checked at the end of the 8th course. PI-PLC beta1 gene expression was evaluated on peripheral blood samples from patients at baseline, and monthly until the 8th cycle of AZA administration. Results. Between September 2008 and February 2010, 32 MDS patients with IPSS risk Low- or Int-1 were enrolled into the study. Most patients had a normal karyotype (63%) by metaphase cytogenetics, were BRC transfusion-dependent (81%), receiving a median of 4 units/mo, and were previously unresponsive to treatment including ESAs (69%). Twenty-six patients (81%) completed the treatment plan (8 cycles). The Overall Response Rate after the 8th cycle was 58% (15/26 pts) whereas 42% of patients maintained a stable disease; no patient progressed towards a high-risk MDS or AML. Five (19%) patients reached a complete remission whereas 10 (38%) achieved an hematological improvement. Transfusion independent was achieved in 8/26 patients (31%). The median duration of the response was 10 months; five patients maintain their response, that is CR in 2 cases (+24 and +30 months) and HI-E in 3 cases (+14, +25,+26 months) without any treatment or supportive therapy. No clinical or hematologic factor showed a correlation with the response. PI-PLCbeta1 gene expression was quantified in MDS patients at baseline and during AZA treatment. Results were calculated as a percentage ratio of PI-PLCbeta1 expression during AZA treatment compared to baseline. All but one patients (14/15) who achieved an hematologic response during treatment with AZA showed a statistically significant increase in PI-PLCbeta1 mRNA expression (P

Definite spectra of point mutations in the Bcr-Abl kinase domain (KD) are known to confer resistance to the tyrosine kinase inhibitors (TKIs) imatinib (IM), dasatinib (DAS) and nilotinib (NIL) in Philadelphia-positive (Ph+) leukemias. Until the recent advent of next-generation sequencing (NGS) technologies, no method was available that could conjugate high sensitivity, possibility to screen for any known or unknown sequence variation and accurate quantitation of mutated subclones. Because of these key technical limitations, the clinical relevance of early detection of small mutated subclones could never be fully elucidated. We have set up and optimized a Bcr-Abl KD mutation screening assay taking advantage of the Roche 454 NGS technology on a GS Junior instrument, that allows parallel pyrosequencing of a hundred thousand clonally amplified DNA molecules of 400 bp average length. We have thus designed 4 partially overlapping amplicons covering the whole KD of the Bcr-Abl transcript (a.a. 240–520) to be generated by nested RT-PCR using sequence-specific primers conjugated with multiplex identifiers – allowing to pool and sequence different samples from one or multiple patients (pts) in a single run. The assay proved capable to identify, characterize and quantitate sequence variations in samples from pts already known to harbour mutations as assessed by conventional Sanger sequencing with 100% concordance. Given that the number of sequence reads generated in each run is relatively constant, the lower detection limit is inversely correlated with the number of samples that can simultaneously be analyzed. Sensitivity could thus be easily modulated – analyzing a single sample per run allowed to routinely achieve lower detection limits ranging between 0.02 and 0.05% (that is, to detect as little as 2 to 5 mutated Bcr-Abl transcripts in a total of 10,000 Bcr-Abl transcripts). With this target sensitivity, we first retrospectively analyzed samples collected at the time of diagnosis from selected chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) cases known to have developed a T315I mutation 3 to 9 months after TKI start. We found that the T315I could not always be traced back to the time of diagnosis. We also found that, both in CML and in Ph+ ALL pts, several low-level mutations (in the range of 0.05 to 1%) with apparently no clinical relevance (e.g., never reported in association with TKI resistance) were routinely detectable – and appeared not to be predictors of a higher level of genetic instability. More interestingly, when used for mutation monitoring of de novo Ph+ ALL pts enrolled in clinical trials with DAS or NIL, NGS could highlight emerging resistant clones far earlier than D-HPLC or conventional sequencing. Also, some IM-resistant CML and Ph+ ALL pts who had been scored as unmutated by conventional sequencing could be shown by NGS to harbour DAS- or NIL-resistant mutations at 1–10% level – a phenomenon that is known to happen because of the clonal deselection resulting from the temporary lack of any TKI selective pressure in the interval between IM discontinuation and DAS or NIL start. Analyses will be presented in detail. We can conclude that: 1) the 454 NGS technology on the GS Junior instrument is a reliable and cost-effective method to perform Bcr-Abl KD mutation screening of Ph+ leukemia pts – with a target sensitivity of 0,1%, 4 samples can simultaneously be analyzed with costs comparable to those of conventional Sanger sequencing and the advantage to quantitatively follow the dynamics of mutated subclones over time; 2) in line with a recent report, small mutated subclones with apparently no clinical relevance can be detected both in newly diagnosed CML and Ph+ ALL pts before TKI start – which further underlines that mutation screening of pts at diagnosis is uninformative, if not misleading; 3) during TKI therapy, the high sensitivity of NGS allows to detect emerging Bcr-Abl mutant subclones earlier than D-HPLC or conventional sequencing; this is particularly relevant: i) for mutation monitoring of Ph+ ALL pts, that are highly prone to develop resistance and mutations while on TKI therapy, and ii) in IM-resistant CML and Ph+ ALL pts, where NGS may uncover the presence of DAS- or NIL-insensitive mutations, and may thus help choosing the second-line therapeutic strategy most likely to be successful.