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We present a case of a patient with a three-month history of peripheral blood cytopenia without a confirmed diagnosis of myelodysplastic syndrome, who developed a favourable-risk acute myeloid leukemia (AML), according to the European Leukemia Net (ELN) criteria. The patient achieved a complete remission with incomplete platelet recovery (CRi) after induction. The patient achieved the morphological CR after the first consolidation and completed the first-line treatment with a syngeneic stem cell transplantation (SCT). A disease relapse occurred after one year of CR (blast cell count in the bone marrow 15%), and the patient was offered a haplo-SCT, which he refused due to personal reasons. In this paper, we discuss the interplay between clinical and biological risk factors in non-high-risk AML patients and speculate that some old clinical risk factors (e.g., age of the patient, achievement of CR after induction, and previous history of myelodysplastic syndrome) may still impact on the treatment decision algorithm of some of these patients.

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

Interferon α (IFNα) prolongs survival of CML patients achieving CCyR and potentially synergizes with TKIs. We report on the molecular status and long term outcome of 121 patients who were treated in Italy between 1986 and 2000 with IFNα based therapy and who obtained CCyR. After a median follow up of 16.5 years, 74 (61%) patients were switched to standard imatinib: 48 (65%) lost the CCyR on IFNα, and 36 (75%) are alive and in CCyR; 26 (35%) were switched to imatinib when they were still in CCyR on IFNα, and all 26 are alive and in CCyR. Forty-seven patients (39%) were never switched to imatinib: 24 (51%) continued and 23 (49%) discontinued IFNα, respectively, and 39/47 (83%) are alive and in CCyR. At last follow-up, the BCR-ABL transcripts level was available in 96/101 living patients (95%) The BCR-ABL:ABL ratio was between 0.1 and 0.01% (MR(3.0) ) in 17%, and less than 0.01% (MR(4.0) ) in 81% of patients. No patient was completely molecular negative (MR(4.5) or MR(5.0) ). The OS at 10 and 20 years is 92 and 84%, respectively. This study confirms that CCyR achieved with IFNα and maintained with or without imatinib or any other therapy significantly correlates with long term survival in CML patients who mostly have MR(4.0) . Complete molecular response (MR(4.5) or MR(5.0) ) seems to be unnecessary for such a long survival. This study further supports development of studies testing the clinical effect of the combinations of TKIs with IFNα.

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.

The activation of Chk1 has been demonstrated to influence response to chemotherapy in patients with BCR-ABL-positive chronic myeloid leukemia patients (Nieborowska-Skorska et al, Cell Cycle 2006). Chk1 and Chk2 are serine threonine kinase activated by different insults leading to DNA damage and involved in DNA repair and cell cycle control. Since, their role has not been established in BCR-ABL+ ALL, we investigated the expression of Chk1/Chk2 in blast cells from adult BCR-ABLpositive ALL patients (n=48) compared with normal bone marrow precursor cells (n=9) using the BioMark instrument (Fluidigm) and Fluidigm Dynamic Array 48 x 48. The expression of Chk1 but not Chk2 was higher (p=0,0005) in BCR-ABL+ leukemia patients (median value 0,69) compared with normal samples (median value 0,27). In order to better understand the biological role of Chk1 in ALL, we included in the analysis different B (BCR-ABL-positive: BV-173 and SUP-B15; BCR-ABL-negative: REH, NALM-6 and NALM-19) and T (MOLT-4, RPMI-8402 and CCRFCEM) leukemia cell lines. The expression of Chk1 was very homogeneous among the cell lines, with no difference between BCR-ABL-positive or negative cells. Since Chk1/2 inhibitors are currently available, we investigated the efficacy of Chk1/2 inhibition by PF-0477736 (Sigma) both as single agent and in combination with tyrosine kinase inhibitors (TKIs), imatinib and nilotinib, in BCR-ABL+ leukemia cell lines. Results showed that the combination (TKIs + Chk1/2 inhibitor) is more effective than the single inhibitors in inducing cell death. In conclusion, adult ALL patients show an higher expression of Chk1 compared with normal bone marrow precursor cells. This abnormal expression could be found also in different B-/T-ALL cell lines, suggesting that the inhibition of Chk1 could be a target for new therapeutic strategies in ALL.

Introduction. Chk1 and Chk2 are serine/threonine kinases that play a critical role in determining cellular responses to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. We explored the cellular effects of single-agent inhibition of Chk1/2 by PF-0477736 and its potential use as a therapeutic strategy for the treatment of B-ALL. Methods. Cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Results. BCR-ABL1-positive (BV-173, SUPB-15) and negative cell lines (NALM6, NALM19, REH) were incubated with increasing concentrations of PF-0477736 (0.005-2 M) for 24, 48 and 72 hours. Inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1-1.5 μM, with BV-173 being the most sensitive, while NALM6 the most resistant. All cell lines were TP53 wild-type, CDKN2A deleted. Consistent with the viability results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in all cell lines. Functionally, PF-0477736 decreased the inhibitory phosphorylation of Cdc25c Ser216 which is inactivated by Chk1 to prevent mitotic entry and increased the number of H2AX foci, a markers of DNA damage, that culminated in a proportion of cells developing intense staining for H2AX together with nuclear morphological characteristics of apoptosis as demonstrated by immunofluorescence analysis. The efficacy of PF-0477736 was thereafter confirmed in primary blasts from 11 B-ALL patients. Based on the viability results, three groups of patients were identified: very good responders, 46% (IC50: 0.1-0.5 μM at 24 hours); good responders, 36% (IC50: 0.6-1 μM at 24 hours); poor responders, 18% (IC50 > 1 μM at 24 hours). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis was performed on treated cell lines and their untreated counterparts (DMSO 0.1%) after 24 hours. Consistent with a specific Chk1- mechanism of action, treatment resulted in differential expression (p < 0.05) of genes involved in apoptosis and cell cycle (e.g. CEBPB, CUL1, Histone H1-H2A, 2B family clusters) and DNA damage (DDIT3, GADD34 and GADD45a), suggesting that PF-0477736 contributes to accumulation of DNA damage and subsequent apoptosis in B-ALL cells. Conclusions. For the first time we demonstrated the efficacy of PF-0477736 in vitro models of B- ALL, suggesting that single-agent Chk1/2 inhibition may be further evaluated in clinical trials. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, FIRB2006, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer.

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.

Deletions of the 9p21 chromosomal region are frequent events for the development of a variety of cancers, including solid tumors and hematological malignancies, such as childhood ALL. This region contains the 40-kb region encoding the p16INK4a/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15INK4b/CDKN2B, all of which encode critical factors for the regulation of cell cycle and/or apoptosis. In order to explore the frequency and size of deletions affecting the 9p21 locus in adult BCR-ABL1-positive ALL, to determine the main mechanism of inactivation and to investigate the influence on the prognosis, 112 patients were analyzed: 82 (73%) de novo cases, 11 (10%) unpaired relapse cases, 19 (17%) diagnosis-relapse matched samples. The median age was 53 years (range: 18–76) and the median blast percentage was 90% (range, 18–99). Affymetrix single nucleotide polymorphism (SNP) arrays (GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0) were used to identify at a high resolution copy number changes on 9p21. PCR amplification and mutation screening of each exon by cloning and subsequent sequencing were also performed. Moreover, gene-expression profiling was performed (Affymetrix Human Exon 1ST Array) to draw a specific signature associated with deletions. At diagnosis, CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 24% of patients, respectively. Deletions were monoallelic in the majority of cases (72%) with a median of 1,012 kb in size (range, 2.8–31,319 kb). In some of them, 43%, the minimal overlapping region of the lost area spanned only the CDKN2A/ARF/CDKN2B gene locus, but more often (57%) the loss was considerable larger and extended sometimes over the entire short arm eliminating a large number of genes. In contrast, cases with bi-allelic inactivation were 28% with the majority of deletions (75%) limited to CDKN2A/ARF/CDKN2B genes. FISH analysis was performed using three different BAC clones, but since they overlooked microdeletions we only appreciated a mild fluorescent signal reduction. In order to assess whether 9p21 loss is responsible for progression, the genomic status of this locus was assessed at the time of relapse and an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06) was found. Functionally, deletions led to a strong down-regulation at the transcript level of CDKN2A/ARF (p= 0.0005), as demonstrated by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation, South San Francisco, CA) which enables to perform TaqMan nano-reactions at high sensitivity. Finally, the mutation screening performed on each exon of ARF, CDKN2A and CDKN2B genes showed that the 9p21 locus is rarely affected by point mutations with only the identification of the missense D146N in the CDKN2A exon 2. Furthermore, 2 mutations were found in the 5′UTR of CDKN2A [UTR A 21965011 (33%), UTR G/A 21965011 (47%), UTR C/T 21964875 (3%)] and a variation, known as SNP was found in the exon 2 (rs3731249). Finally, 2 SNPs were found in the 3′ UTR region of CDKN2A/ARF (rs11515 and rs3088440). The role of the mutations identified in the UTR is not yet defined, but the comparison with the germline material from saliva samples showed that these mutations were inherited. Since the prognostic relevance of CDKN2A/ARF deletions is still controversial in literature, we addressed this issue in our study cohort, finding out that deletions in this locus are significantly associated with poor outcome both in terms of disease free-survival (p=0.003) and cumulative incidence of relapse (p= 0.004). In conclusion, the inactivation of the tumor suppressor genes CDKN2A/ARF is a frequent event in Ph+ ALL. The main mechanism of inactivation is represented by genomic deletions, whereas missense mutations are rare. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker, impairing disease free-survival and cumulative incidence of relapse. Novel treatment strategies targeting the ARF-MDM2-p53 pathway may be effective in this subset of patients and in vitro studies are ongoing to confirm this hypothesis.