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Abstract 4628 Background. Tribbles homolog gene 2 (TRIB2) is a pseudokinase gene belonging to a three member gene family. Tribbles gene was first identified in Drosophila melanogaster where is involved in the regulation of morphogenesis and mitosis. Likewise, mammalian homologous genes of Tribbles (TRIB1, TRIB2, TRIB3) promotes the degradation of specific transcription factors and interacts with several cell signaling mediators and modulators. Recently it has been demonstrated that TRIB2 was able to induce AML in bone marrow transplanted mouse by inducing C/EBPα proteasome-dependent degradation. Moreover, gene expression profiles data from AML patients revealed that patients carrying C/EBPα mutations clustered with those patients with up-regulated TRIB2.Aims. To test the hypothesis that TRIB2 expression was related to a differentiation degree in AML, we evaluated several AML cases based on FAB cytotype. Patients and Methods. We performed quantitative Real Time-PCR (qRT-PCR) analysis on AML patients (15 AML-M0, 14 AML-M2, 5 AML-M3, 2 M4 and 3 M5b bone marrow aspirate samples) to measure the expression level of TRIB2 in different FAB AML subtype. Four healthy bone marrows were used as reference samples. qRT-PCR was conduct using SYBR green chemistry and specific primers for TRIB2 transcript and for two housekeeping genes (B2M and IPO8) previously tested for their stability and efficiency. All AML sample were tested by conventional cytogenetic analysis and by FISH with TRIB2 specific probe. We performed, also, methylation analysis of a CpG island located in the TRIB2 promoter region by methylation sensitive restriction enzyme (MSRE) and qPCR. Briefly, DNA samples were digested with MSRE and were quantified by qPCR using specific primer pairs surrounding restriction enzymes recognition sites. Results. qRT-PCR experiments revealed that TRIB2 expression was higher (from 3 to 20 fold) in AML-M0 respect to the AML-M2 (p= 0.02), AML-M3 (p=0.01), AML-M5 (p=0.006) FAB subtypes, and references (p=0.003), respectively. Moreover, the AML-M0 cases showed a TRIB2 overexpression compared to that observed in the two AML-M4 cases (about 4 fold changes) but this difference there was not statistically significant probably because of M4 cases paucity in our series. Therefore qRT-PCR displayed a progressive decreasing TRIB2 expression along FAB subtypes. Conventional cytogenetic and FISH analysis did not show any kind of rearrangement involving TRIB2 gene. MSRE experiments revealed an higher methylation degree of a CpG island located in the TRIB2 promoter region in AML-M0 cases respect to the others FAB subtypes; moreover, methylation was significantly correlated with TRIB2 expression (r = 0.9; p = 0.0002). Conclusions. Our data showed that the TRIB2 gene overexpression was associated to AML-M0. In our cases TRIB2 dysregulation was not due to gene amplification as in other cancers. Surprisingly we observed that AML-M0 samples showed an higher methylation degree in the TRIB2 promoter region. Usually methylation cause gene silencing but our results showed that TRIB2 promoter region methylation was associated to the overexpression of the gene. Maybe methylation inhibits binding of some unknown transcriptional repressor as already seen for hTERT gene in others tumors cells. In conclusion, our data revealed that TRIB2 dysregulation seems to be linked to AML-M0 phenotype. Further studies are needed to clarify the reasons for this association. Disclosures: No relevant conflicts of interest to declare.

Abstract 2270 An update of the European LeukemiaNet criteria for monitoring response of chronic myeloid leukemia patients was recently published and provisional criteria to evaluate patients during second generation TKI therapy after resistance to imatinib were proposed. In our study we retrospectively tested these criteria in a large series of CML patients resistant to imatinib further treated with second generation TKIs with the aim to analyze the outcome of suboptimal response and failure patients compared to those with optimal response and to validate the provisional criteria for monitoring response. One hundred twenty-seven CML patients resistant to imatinib were collected from 6 different Italian hematologic centers. There were 66 males and 61 females, median age 54 years (range 25–80). Twenty-seven patients were in late chronic phase after IFN resistance. Ninety-seven patients received second-generation TKI after acquired resistance, whereas 30 patients had primary resistance. We found that at different time points (3, 6 and 12 months), patients classified as failure showed significantly worse 2-year overall survival (OS), progression-free survival (PFS) and event-free survival (EFS) than sub-optimal and optimal response patients. At 3 months, “failure” patients, had an OS of 83% compared to 86% of sub-optimal and 97% of optimal response patients (p=0.001); PFS was 77% for failure patients compared to 92% and 99% for sub-optimal and optimal response patients, respectively (p=0.001), whereas EFS was 41% for failure vs 59% for sub-optimal (p=0.001) and 85% and optimal response patients, respectively (sub-optimal vs optimal p Disclosures: No relevant conflicts of interest to declare.

Background The t(9;22)(q34;q11), generating the Philadelphia (Ph) chromosome, is found in more than 90% of patients with chronic myeloid leukemia (CML). As a result of the translocation, the 3' portion of the ABL1 oncogene is transposed from 9q34 to the 5' portion of the BCR gene on chromosome 22 to form the BCR/ABL1 fusion gene. At diagnosis, in 5-10% of CML patients the Ph chromosome is derived from variant translocations other than the standard t(9;22). Results We report a molecular cytogenetic study of 452 consecutive CML patients at diagnosis, that revealed 50 cases identifying three main subgroups: i) cases with variant chromosomal rearrangements other than the classic t(9;22)(q34;q11) (9.5%); ii) cases with cryptic insertions of ABL1 into BCR, or vice versa (1.3%); iii) cases bearing additional chromosomal rearrangements concomitant to the t(9;22) (1.1%). For each cytogenetic group, the mechanism at the basis of the rearrangement is discussed. All breakpoints on other chromosomes involved in variant t(9;22) and in additional rearrangements have been characterized for the first time by Fluorescence In Situ Hybridization (FISH) experiments and bioinformatic analyses. This study revealed a high content of Alu repeats, genes density, GC frequency, and miRNAs in the great majority of the analyzed breakpoints. Conclusions Taken together with literature data about CML with variant t(9;22), our findings identified several new cytogenetic breakpoints as hotspots for recombination, demonstrating that the involvement of chromosomes other than 9 and 22 is not a random event but could depend on specific genomic features. The presence of several genes and/or miRNAs at the identified breakpoints suggests their potential involvement in the CML pathogenesis.

Abstract 3261 Poster Board III-1 A crucial role of segmental duplications (SDs) of the human genome has been demonstrated in chromosomal rearrangements associated with several genomic disorders. Limited knowledge is yet available on the molecular processes resulting in chromosomal rearrangements in tumors. The t(9;22)(q34;q11) causing the 5'BCR/3'ABL gene formation has been detected in more than 90% of chronic myeloid leukemia (CML) cases. Some years ago, a 76-kb duplicon was reported, closely located to both ABL and BCR genes which are involved in the t(9;22)(q34;q11) translocation associated with CML. However, the exact role of this duplicon in mediating the t(9;22) rearrangement remained mostly speculative. In 10–18% of CML patients genomic deletions were detected on der(9) chromosome next to translocation breakpoints. The molecular mechanism triggering the t(9;22) and deletions on der(9) is still unknown. Our study presents an experimental evidence of the involvement of SDs in the genesis of the t(9;22) translocation in CML and in the occurrence of genomic deletions on der(9) chromosome. We identified 71 (17%) cases with der(9) deletions by FISH screening of 416 CML patients at diagnosis. Fine-mapping of deletions was performed using appropriate bacterial and Phage P1-derived artificial chromosome clones. The deletions sizes were heterogeneous, ranging from 230 Kb to 12.9 Mb on chromosome 22 and from 260 Kb to 41.8 Mb on chromosome 9. The mapping of all breakpoints revealed an evident breakpoints clustering, on both chromosomes 9 and 22, in two regions of about 2 Mb in size. Indeed, these regions contained the breakpoints detected in 54 out of 60 (90%) patients bearing chromosome 9 deletions and in all patients with chromosome 22 sequences loss. Bioinformatic analysis of chromosome 9 and chromosome 22 genomic regions involved in the deletions was performed to search for features that could correlate with the breakpoints clustering. To this aim, the breakpoint regions were subdivided into 250 Kb intervals. The most striking result was the fact that both clusters contain the above reported 76-kb duplicon, shared by chromosome 9 and 22 (SD_9/22). The SD_9/22 is the only duplication located inside the breakpoints clustering region on chromosome 9, whereas the chromosome 22 clustering region harbors several duplications. A remarkable feature of the chromosome 9 clustering region was the high frequency of Alu repeats. The mean Alu frequency overall on chromosome 9 is 10.8%, whereas the average Alu content on this cluster is 31.3%. Accordingly, as expected, the content in LINE sequences of the region was relatively low (average overall on chromosome 9: 21.2%, as opposed to 8.7% on the cluster region). Gene distribution analysis of chromosome 9 and 22 showed that both SD_9/22 map inside gene-poor regions, of about 460 Kb and 250 Kb in size, respectively. To corroborate the observations on the distribution of SDs and Alu/LINE repeats, the chromosome 9 and 22 regions surrounding the SD_9/22 were once more divided into 250 Kb segments, positioning the SD_9 and SD_22 as landmarks. A statistically significant negative association was observed between the number of breaks and the distance from SD_9/22, on both chromosomes 9 (p=0.01) and 22 (p=0.006), respectively. The relationship between the breaks and the interspersed repeats revealed, on chromosome 9, a positive linear regression with Alu repeats (p=0.04), and a negative one with LINEs (p=0.04). Very similar conclusions were obtained by comparing the distance from the SD_9 and the Alu (p= 0.03, positive) and LINE distribution (p=0.02, negative). No statistically significant relationship was observed on chromosome 22. In our study the involvement of SDs was proposed to explain the recurrent t(9;22) translocation in CML and the genomic deletions that could accompany the rearrangement. Although the chromosomes 9 and 22 breakpoints clustering regions are quite large, the strong non-randomness of SD_9/22 location and the genomic features identified in our work suggest that the chromosomal segments near the ABL and BCR genes are brought together by an active process, facilitating recombination. At the light of these findings, the analysis of secondary non-recurrent events could represent a new methodological approach able to identify architectural elements involved in the occurrence of recurrent primary rearrangements in human neoplasia. Disclosures: No relevant conflicts of interest to declare.

Abstract 2211 Poster Board II-188 Dasatinib is a 2nd generation tyrosine-kinase inhibitor active in CML patients resistant or intolerant to Imatinib; at present there is no data on its toxicity and efficacy in unselected elderly patients. To highlight this issue, 97 patients treated with Dasatinib when aged > 60 years were retrospectively evaluated from 16 Italian Centers on a “real-life” basis, including all patients treated at each Center independently from enrolment or not in controlled clinical trials.There were 52 males and 45 females, median age at Dasatinib start was 69.5 years (IR 65.0 – 73.3), Sokal Risk at diagnosis was low in 26 patients, intermediate in 37, high in 15 and not valuable in 19. Forthy-five patients (46.4%) were primarily resistant, 11 (11.4%) were intolerant and 41 (42.2%) had secondary resistance to Imatinib; all patients were in CP when Dasatinib was started. Median time from diagnosis to Dasatinib treatment was 85.0 months (IR 44.8 – 120.0); 53/97 patients (54.6%) had been pretreated with IFN ± Ara-C before Imatinib, all patients received Imatinib at standard dose (400 mg/day) followed in 50/97 (51.5%) by increased dose (600 – 800 mg/day) with an overall median period of Imatinib treatment of 48.6 months (IR 26.9 – 67.0). In addition, 28/97 patients (28.8%) received other 2nd line treatment (10 Nilotinib, 14 HU +/- other drugs, 3 Imatinib + HU or IFN and 1 allogeneic transplant) before Dasatinib. Starting dose of Dasatinib was 140 mg/day in 47 patients, 100 mg/day in 44 patients and ≥ 50 mg/day in 6 patients, respectively. After a median period of treatment of 15.6 months (IR 7.6 – 23.0) all patients were evaluable for toxicity; on the whole, grade 3 – 4 hematological and extra-hematological toxicities were reported in 36/97 (37.1%) and 27/97 (27.8%) patients, respectively. A grade 3 – 4 hematological toxicity occurred in 25/47 (53.1%) patients receiving 140 mg as compared to 10/44 (22.7%) patients receiving 100 mg (p 60 years resistant/intolerant to Imatinib; in particular, when employed at the current recommended dose of 100 mg/day, it is very effective and has a favourable safety profile also in heavily pretreated elderly subjects. Disclosures: Vitolo: Roche: . Pane:Novartis: Research Funding; Ministero dell'Università/PRIN: Research Funding; Regione Campania: Research Funding; Ministero della Salute/Progetto integrato Oncologia: Research Funding.

MicroRNAs (miRNAs) are small, single stranded non-coding RNAs, 19–24 nucleotides long, involved in crucial biological processes, including differentiation, apoptosis and proliferation. Recent evidence indicates that miRNAs may play an important role in tumorigenesis; changes in miRNAs expression level were identified in many types of human hematological and solid malignancies. To date, several papers have reported the occurrence of genomic deletions flanking the breakpoint on der(9)t(9;22) in 10%–18% of patients with chronic myeloid leukemia (CML). The most probable consequence of der(9) deletions is the loss of tumour suppressor genes, conferring a proliferative advantage to the Philadelphia-positive clone. On the other hand, two miRNAs, namely miR219-2 and miR-199b, are found to map centromeric to the ABL gene within the chromosomal region at 9q34 that is frequently lost in CML patients with der(9) deletions. In this study, we investigated the loss of miR-219-2 and miR-199b by fluorescence in situ hybridization (FISH) analysis with specific bacterial artificial chromosome (BAC) probes in 68 CML cases bearing der(9) deletions. We further evaluated miR-219-2 and miR- 199b expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) experiments in cases showing deletions of at least one of these miRNAs. Depending on RNA sample availability, miRNAs expression level was evaluated in 7 and in 5 CML cases with miR-219-2 and miR-199b deletions, respectively. Statistical analysis of the relative expression results was performed by the Relative Expression Software Tool (REST). To explore the predicted miR-199b target genes, the miRGen targets database (http:// www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi) was queried; this interface provides integrated data of four widely used target prediction programs (miRanda, PicTar, TargetScan, DIANA-microT). FISH experiments revealed the loss of miR-219-2 and miR-199b in 17 (25%) and 10 (15%) out of 68 patients. The miR-199b expression study showed a downregulation in the analyzed group of 5 CML cases with miR-199b deletion as compared to a pool of 10 patients without deletions. The expression level of the miR-199b was 0.279 and the difference between the two groups was statistically significant (p= 0.028). On the contrary, the miR- 219-2 analysis did not reveal a detectable expression level in the examined patients. There were 26 predicted miR-199b target genes, involved in several biological processes such as signal transduction (Protein phosphatase inhibitor 2, PPP1R2), regulation of transcription (Hepatic leukaemia factor, HLF), chromosome organization and biogenesis (Zinc finger protein 238, ZNF238), cell proliferation (Mitogen-activated protein kinase 11, MAP3K11) and DNA repair (UV excision repair protein RAD23 homologue B, RAD23B). Among the CML patients evaluable for the response to the treatment, all cases with the miR-199b deletion were resistant to IFN-a and imatinib therapy. In conclusion, our data demonstrate a crucial role for miR-199b in CML cases bearing der(9) deletions. This miR-199b downregulation could influence the expression level of different target genes modifying important cellular pathways. Further analysis of miR-199b target genes will be needed to shed light on the link between miRNAs and CML.

Deletions on der(9) are associated with chronic myeloid leukemia(CML) in 15–18% of cases. To date, the biological significance of this genomic loss in the pathogenesis of CML is unknown. The most plausible hypothesis is that the loss of a tumor suppressor gene may confer a proliferative advantage to the Philadelphia-positive clone. On the other hand, it has now become evident that microRNAs (miRNAs) play an important regulatory role in some hematological malignancies. To investigate the presence of miRNAs within the genomic regions lost on der(9) we analyzed 60 CML patients with der(9) deletions. Methods. Genomic characterization of the deleted sequences was performed by fluorescence in situ hybridization (FISH) using a contig of DNA clones; the miRBase (http://microrna.sanger.ac.uk/) was queried to assess the presence of miRNAs in the der(9) deleted genomic regions. FISH experiments showed that the genomic loss on der(9) of the 9 (centromeric to ABL) and 22 (telomeric to BCR) chromosome sequences ranged from 260 Kb to 54 Mb and from 230 Kb to 12.9 Mb, respectively. Consultation of the miRBase revealed that in 16 (27%) patients there was loss of miRNAs mapping on chromosome 9 whereas no known miRNAs were mapped on the deleted genomic sequences belonging to chromosome 22. Moreover, 4 cases with a complex t(9;22) rearrangement and der(9) deletions showed loss of the miRNAs sequence also on the third derivative chromosome (4p16, 7p14, 13q14, and 11q13, respectively); among them, only in one case the loss of miRNAs on the third derivative was not associated with the miRNAs deletion mapped on chromosome 9. The most recurrent miRNAs deleted on der(9) were mir-219–2 (deleted in 100% of cases) and mir-199-b (lost in 67% of cases). It is noteworthy that mir-219–2 neighbors and overlaps CpG-islands, suggesting a potential role of this miRNA in CpG-island methylation. Experimental studies indicate that miRNAs can function as tumor suppressor genes or as oncogenes. In fact, in chronic lymphocytic leukemia associated with del(13)(q14) it has been demonstrated that the miRNAs loss can induce downregulation of the antiapoptotic BCL-2 protein. The novel evidence that deletions on der(9) in CML are associated with miRNAs loss may shed new light on the significance of genomic sequences loss. Further studies are needed since it is known that some microRNAs may have as many as a few thousand targets, so prediction algorithms and strategies allowing large-scale screening of multiple target genes are required.

It is known that the receptor activator of nuclear factor kB (RANK) is only identified on progenitor and mature osteoclasts and in dendritic cells. A recent report (Blood107:4334, 2006) described a dose-dependent decrease of RANK expression in osteoclasts when cultured in the presence of imatinib; these data were confirmed in adult mice treated with imatinib, in which a decrease in osteoclasts activity was demonstrated. Therefore, it is speculated that imatinib may have a potential antiresorptive effect. We evaluated bone marrow RANK molecular expression in patients affected by chronic myeloid leukemia (CML) receiving imatinib therapy, to verify the effect of the tyrosine kinases inhibitor on RANK expression. The study included 13 CML patients (7 males, 6 females, median age 37 yrs). Six were treated with Imatinib at standard dosage (400 mg) and the remaining 7 at higher doses (> 400 mg). The median duration of imatinib therapy was 28 months (range 13 – 44 months). The RANK gene expression level was determined by Real-Time PCR using 1X Platinum SYBR Green qPCR SuperMix-UDG. The RANK gene expression level variation was estimated by comparing the values of 2−Δ Δ Ct obtained in CML patients at diagnosis and during imatinib treatment. For each of the tested cases the sample at diagnosis was utilized as calibrator and 28S rRNA was used as reference gene. Eleven (85%) patients showed an upregulation of RANK, the gene expression fold increase ranging from 1.17 to 15.88 as compared to the value at diagnosis; all patients treated with high-dose imatinib showed a RANK overexpression, the fold increase ranging from 1.57 to 15.88. Although the median RANK expression fold increase observed in the group of patients treated with high-dose imatinib was higher than in the cases taking standard dosage (2.47 versus 1.34 fold increase, respectively), this difference was not statistically significant. There was no association between the RANK expression level and the duration of imatinib treatment. Our study reveals that imatinib mesylate is accountable for the molecular overexpression of the RANK gene in CML patients. These data seem to be in conflict with those previously reported (Blood2006;107: 4334). It is possible that the RANK overexpression which we observed was related to the immune (i.e. dendritic cells) and not the bone cells (i.e. osteoclasts). In this regard, a new type of cell has recently been described in mice, that may account for the immune effect of imatinib; the new cell, which seems to be a cross between a dendritic cell and a natural killer cell, has been called an “interferon-producing killer dendritic cell” (Nat Med2:214, 2006; Nat Med2:207, 2006). These studies suggest a new mechanism by which imatinib might exert an antitumor effect, apart from its direct action on chronic myeloid leukemia cells. Therefore, in this context, the RANK upregulation observed in our study could be an expression of the immune effect of imatinib. Further studies are needed to verify if the RANK gene overexpression mediated by imatinib could affect the molecular and cytogenetic response in CML patients.

The receptor activator of the nuclear factor kB ligand (RANKL) is a key molecule in the pathogenesis of malignant osteolytic lesions in multiple myeloma or bone metastases in prostate and breast cancer. Inhibition of RANKL is therefore a promising new therapy for controlling bone loss and pain in bone tumors and associated osteolysis. Changes in bone and mineral metabolism developing in a proportion of patients taking imatinib for either chronic myeloid leukemia (CML) or gastrointestinal stromal tumors have previously been described. The aim of our study was to verify in CML patients the effect of imatinib on RANKL bone marrow molecular expression. The study included 13 CML patients (7 males, 6 females, median age 37 yrs). Six were treated with imatinib at standard dosage (400 mg) and the remaining 7 at higher doses (> 400 mg). The median duration of imatinib therapy was 28 months (range 13 – 44 months). The RANKL gene expression level was determined by Real-Time PCR using 1X Platinum SYBR Green qPCR SuperMix-UDG. Variations in RANKL gene expression level were estimated by comparing the values of 2−Δ Δ Ct obtained in CML patients at diagnosis and during imatinib treatment. For each of the tested cases the sample at diagnosis was utilized as calibrator and 28S rRNA was used as reference gene. Eight (62%) patients showed a downregulation of RANKL gene expression, the fold decrease ranging from 0.01 to 0.47 as compared to the value at diagnosis; among these 8 patients, 6 (75%) were treated with imatinib at standard dosage. Instead, 5 (38%) patients showed an upregulation of RANKL, the gene expression fold increase ranging from 1.43 to 4.94 as compared to the gene level observed at CML onset; all these 5 patients were treated with high doses of imatinib. There was no association between RANKL expression and the duration of imatinib treatment. These findings suggest that imatinib mesylate could control RANKL expression. At standard dosage, imatinib can downregulate RANKL production. The RANKL upregulation observed in CML patients treated with high doses of imatinib could be explained by the possible activation of the immune system cells, such as T and B cells, induced by the tyrosine kinases inhibitor. Further studies are needed to verify the minimal effective dose of imatinib on RANKL expression, with the aim of administering it as an antiosteolytic agent in such diseases as osteoporosis, metastatic bone disease, and multiple myeloma.

Extramedullary infiltration (EMI) of malignant myeloid precursor cells in acute myeloid leukemia (AML) may occasionally be a presenting clinical symptom at onset and may develop at any site in the body but most commonly in the gum, skin, central nervous system (CNS) and soft tissue. There is controversy about the prognostic significance of extramedullary disease in AML. The present study examines the incidence, the biological features and prognostic significance of EMI at diagnosis in adult patients with AML. From January 1997 to December 2004, 213 untreated patients with de novo AML were studied. According to Grimwade et al, 14 patients were in the favorable-risk group, 159 in the intermediate-risk group, 25 in the poor risk group and in 15 the karyotype was not available. All patients had been treated with induction therapy according to the GIMEMA protocols including cytarabine, etoposide and idarubicin (15 pts), or mitoxantrone (15 pts) or daunorubicin (183 pts). Of 213 cases with de novo AML, 29 (14%) had EMI at diagnosis. Ten patients (34.8%) had skin infiltrates, 12 (41.4%) had gum hypertrophy, 5 (17.2%) had CNS involvement and 2 (6.9%) had soft tissue infiltration. No significant differences in terms of sex, age median Hb level and platelets count were found between patients with EMI and patients without EMI. The patients with EMI had higher median WBC counts (27 × 109/L) than patients without EMI (8.5 × 109/L) (p=0.05). The patients with EMI had a higher incidence of the M4/M5 FAB subtype (62%) than patients without EMI (27.4%) (p=0.005). Cytogenetic analysis was performed in patients with and without EMI; none of the abnormal cytogenetic findings was associated with EMI. We evaluated the relationship between the AML blasts surface antigen expression and EMI; the association between CD56/CD4 and CD56/CD14 was more significantly expressed in patients with EMI (35% and 29%, respectively) than without EMI (10.4% and 6.9%, respectively) (p=0.004, p=0.003). The overall CR rate was 65%; the CR rate was lower in patients with EMI (48.2%) than patients without EMI (76.1%) (p=0.001) and their disease free survival was also shorter (p=0.017); the median duration of CR was 10 and 25 months (range 2–96) in EMI and no EMI group, respectively. Our data show that a high WBC count, M4/M5 subtype, CD56/CD4 and CD56/CD14 expression are associated with extramedullary infiltrates of AML at diagnosis; the presence of EMI adversely affects the complete response rate to induction chemotherapy and the OS rate. Analysis of the clinical and biologic features in a larger series of adult AML patients is needed to evaluate the allocation of this subgroup in a different or more intensive treatment arm.

Chronic myeloid leukemia (CML) is characterized by a reciprocal translocation t(9;22)(q34;q11) that generates a BCR-ABL fusion gene on the Philadelphia (Ph) chromosome. Large deletions adjacent to the t(9;22) breakpoint on the derivative 9 chromosome have now been found, which result in genomic loss at both sides of the translocation breakpoint. We report a FISH study in CML cases bearing deletions on the der(9) chromosome. FISH analysis with specific BAC/PAC clones for the ABL1 and BCR genes was performed on bone marrow cells of 305 CML patients at diagnosis. A set of BAC/PAC probes, belonging to chromosomes 9, 22 was selected according to the University of Santa Cruz (UCSC) database and employed in FISH experiments. We detected der(9) deletions in 56 (18.4%) CML cases. Deletions of chromosome 9 sequences on the der(9) were found in 47 (84%) cases; they ranged from 350 Kb to 41.6 Mb. Chromosome 22 deletions on der(9) were found in 49 (87.5%) of the analysed cases; the deleted chromosome 22 sequences were shorter than the deleted chromosome 9 sequences (ranging from 400 Kb to 12.7 Mb). Although deletions size appeared to be heterogeneous, 2 main breakpoints cluster regions were identified proximally (at about 1.9 Mb) to the ABL and distally (at about 0.7 Mb) to the BCR genes, respectively. In fact, a cluster region of 1.1Mb, delimited by RP11-203J24 and RP11-247A12, was defined on chromosome 9 whereas a 0.8 Mb region, included between CTA-322B1 and RP5-930L11, was identified on chromosome 22. Bioinformatic analysis of breakpoints cluster regions was performed to identify sequence motifs that could mediate the chromosomal rearrangement. The presence of Matrix Association Regions (MARs) and the topoisomerase II consensus was investigated by MAR-Wiz software (http://www.futuresoft.org/modules/MarFinder/); the RepeatMasker program (http://www.repeatmasker.org/) was employed to detect interspersed repeats and low complexity DNA sequences (such as SINE, LINE, and LTR). Potential sequence similarities between breakpoints cluster regions were searched for using Genalyzer software. Sequence analysis revealed that the frequency distribution of MARs, topoisomerase II sites, and repeated elements was no different from the frequency observed in other genomic regions. Genalyzer software did not detect sequence similarities between the 2 clustering regions; however, a 76 Kb duplicon, previously reported in literature, was located at a distance of 500 kb and 550 Kb from the chromosomes 9 and 22 cluster regions, respectively. In conclusion, our analysis revealed that sequence motifs do not seem to be involved in the occurrence of der(9) deletions. We hypothesize that the presence of a duplicon could mediate the juxtaposition of ABL and BCR genes and that a probable open chromatin status of chromosomes 9 and 22 sequences could confer to DNA more vulnerability to double strand breaks, mediating the t(9;22) rearrangement and the occurrence of der(9) deletions.

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of Acute Myeloid Leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes generating a 5′RUNX1/3′CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8. In 1 to 8.5% of AML cases insertions events generating a 5′RUNX1/3′CBFA2T1 fusion gene have been reported, whereas the occurrence of inversions accompanying the t(8;21) has never been observed. We report a screening of 82 AML cases bearing the RUNX1/CBFA2T1 rearrangement detected by RT-PCR; all cases were tested by Fluorescence In Situ Hybridization (FISH) with BAC and PAC clones specific for CBFA2T1 and RUNX1 genes. This analysis allowed us to reveal five cases with ins(21;8), one with ins(8;21), and two with a pericentric chromosome 8 inversion followed by a t(8;21) translocation. A detailed molecular cytogenetic characterization of breakpoints has been performed in all cases. FISH co-hybridization experiments with CBFA2T1 and RUNX1 probes revealed the presence of a functional fusion gene on the der(21) instead of the der(8) chromosome in five cases with ins(21;8); a single fusion signal on the der(8) chromosome was detected in the case with ins(8;21). The use of the same clones in FISH studies showed the presence of a single unexpected fusion signal on the 8p derivative chromosome in addition to faint CBFA2T1 and RUNX1 signals on the long arm of der(8) and der(21) chromosomes, respectively. These results suggested that a pericentric chromosome 8 inversion involving CBFA2T1 gene occurred and that the chromosome 21 was rearranged with the 8p derivative chromosome. Appropriate chromosome 21 and 8 BAC clones were employed to precisely define the size of inserted regions in cases with insertions and the breakpoint on the 8p derivative chromosome in cases showing pericentric chromosome 8 inversion. The insertion size turned out to be very heterogeneous, ranging from a minimum of 2.4 Mb to a maximum of 44 Mb. In both cases with chromosome 8 inversion, the CBFA2T1 gene represents the breakpoint at the chromosome 8 long arm whereas the 8p breakpoint showed different mapping positions in 8p21.3 and 8p21.1, respectively. Our results illustrate that (1) heterogeneous mechanisms can lead to the generation of the 5′RUNX1/3′CBFA2T1 chimeric gene; (2) molecular cytogenetic techniques may identify cryptic chromosomal changes, not detected by conventional cytogenetic analysis; (3) the crucial role of the 5′RUNX1/3′CBFA2T1 fusion gene in leukemogenesis does not depend on its location.

Background and Aim Systemic mastocytosis (SM) is characterized by a heterogeneous spectrum of disease variants with different clinical features and prognosis. In indolent SM (ISM), the clinical course is usually stable and patients (pts) have a normal life expectancy. In contrast, aggressive SM (ASM) and mast cell leukemia (MCL) are characterized by severe organ damage and are fatal forms with short survival times. All forms of SM share the presence of activating mutations in the KIT tyrosine kinase (most frequently, the D816V mutation), which suggests that cooperating events might be responsible for the profoundly different clinical presentation and outcome of advanced SM (ASM and MCL) as compared to ISM. However, no robust studies using genome-wide approaches to scan for additional molecular aberrations in ASM and MCL have been published to date. This prompted us to use exome and RNA sequencing and single nucleotide polymorphism (SNP)-arrays to search for novel (and potentially druggable) molecular lesions. Methods Seven pts with MCL, 6 pts with ASM and 2 pts with ISM were studied using whole exome sequencing (WES) and single nucleotide polymorphism (SNP)-arrays. WES (80x) was performed on a Hiseq 2500 (Illumina). SNP-arrays were done using Cytoscan HD Arrays (Affymetrix). Matched normal/mast cell DNA was analyzed in all but 2 archival MCL cases for whom no germline DNA source was available. RNA sequencing was also performed in 3/7 MCL and 1/6 ASM cases for whom high quality RNA was available. Data analysis and integration was performed according to in-house optimized pipelines. Results SNP-arrays found arm-level copy number (CN) or copy-neutral loss-of-heterozygosity (CN-LOH) events in 5/7 MCL pts (loss of 7p, 8p, 9q, 12p, 13q and CN-LOH at 4q, 18q and 21q). In contrast, ASM and ISM showed only focal CN and CN-LOH events. Overall, a median of 28 (range, 20-78) and 77 (range, 55-132) submicroscopic CN and CN-LOH events, respectively, were detected. The number of events did not significant differ between ISM, ASM and MCL. A total of 54, 102 and 1835 genes were significantly found to be recurrently involved in loss, gain and CN-LOH events, respectively. The average number of nonsynonymous mutations identified by WES was 15 in ISM and 35 in ASM and MCL. Signature analysis showed that the majority of sequence changes were C>T transitions and C>A transversions. Among genes recurrently mutated in ≥2 patients or concurrently identified by WES and SNP-arrays or known to be associated with cancer, some had already been reported to be mutated in SM: ASXL1, TET2, CBL, RUNX1, NRAS, IDH1, SRSF2, SF3B1. Alterations were also found in genes not previously implicated in SM, including RUNX3, MLL2, MLL3, CDC27, TP53BP1, CCND3, NCOR2, BCORL1, ATM, WRN, ARID1B, ARID3B, KDM1B, ARID4A, SETD1A, SETD1B, PRDM1. Pathway analysis showed enrichment of genetic lesions affecting the following cellular processes: PI3K/Akt and MAPK pathways, calcium signaling, chromatin modification, DNA methylation and DNA damage repair. RNA-seq results, filtered against those of a set of normal bone marrow samples, revealed a high number of RNA chimeras involving two adjacent genes in the same transcription orientation and invariably joining the pre-last exon of the upstream gene to the second exon of the downstream gene - a fusion pattern suggestive of intergenic splicing following transcription read-through events. Deletions at the intergenic region was excluded by SNP-arrays for all the predicted transcription-induced chimeras. In addition, in all the 4 pts analyzed, retention of some or all introns in mature transcripts was observed for some genes, including known tumor suppressors (TS) and other key genes like TP53, BAX, ERCC2, WRN, FANCD2, ATR; U2AF2, U2AF1, SRSF6. Conclusions SNP-array and WES analysis of SM revealed a heterogeneous landscape of molecular alterations, although some key cellular pathways were found to be recurrently altered. More interestingly, RNA-seq uncovered perturbation of transcript elongation and splicing in all the advanced SM pts analyzed. Intron retention, in particular, seems to be a novel recurrent mechanism of TS gene inactivation at the posttranslational level in advanced SM. The mechanisms leading to intron retention and their cellular consequences deserve further investigation. Supported by AIRC (project code 16996), progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Cavo:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding. Martinelli:MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau.

PURPOSE: Recent reports of extramedullary disease (EMD) at recurrence in acute promyelocytic leukemia (APL) have raised increasing concern about a possible role of retinoic acid (RA) therapy. PATIENTS AND METHODS: We analyzed the risk of developing EMD localization at relapse in APL patients enrolled onto two consecutive studies of the Gruppo Italiano Malattie Ematologiche dell’Adulto. The studies investigated chemotherapy alone (LAP0389) versus RA plus chemotherapy (AIDA). RESULTS: When all relapse types were taken into account, 94 (51%) of 184 patients and 131 (18%) of 740 patients who attained hematologic remission underwent relapse in the LAP0389 and AIDA studies, respectively (P < .0001). EMD localization was documented in five (5%) of 94 and 16 (12%) of 131 patients (P = .08). Hematologic and/or molecular relapse was diagnosed concomitantly in all but two patients with EMD in the AIDA study. For patients in the LAP0389 and AIDA series, the probability of EMD localization of any type at relapse was 3% and 4.5%, respectively (P = .79), while the probability of CNS involvement was 0.6% and 2% (P = .28). No significant differences were found with regard to mean WBC count and promyelocytic leukemia/retinoic acid receptor-alpha junction type in comparisons of patients with EMD and hematologic relapse. CONCLUSION: APL patients receiving all-trans retinoic acid in addition to chemotherapy have no increased risk of developing EMD at relapse as compared with those treated with chemotherapy alone.

In adult acute myeloid leukemia (AML), a variety of clinical and biological parameters have been examined for their potential value in predicting treatment response. Early response to induction therapy could be an important prognostic factor in this disease.We studied the relationship between reduced blasts in bone marrow aspirate on the 14th day (BMA14th) of induction chemotherapy and treatment outcome in 198 adult AML patients of whom 124 were60 years old (group A) and 74or = 60 years old (group B). Receiver operating characteristic curve analysis was used to assess the prognostic performance of BMA14th. Using the percentages of blasts ofor = 22% andor = 15% as criteria for predicting treatment outcome gave the highest accuracy in terms of sensitivity and specificity in groups A and B, respectively.In group A, of 97 patients with a BMA14thor = 22%, 77 (79%) achieved complete remission (CR), whereas of 27 patients with a BMA14th22%, 22 (81%) were non-responders (NR) (p0.0001). The test sensitivity and specificity were 93.9% and 71.4%, respectively. In group B, of 27 patients with a BMA14thor = 15%, 18 (67%) achieved CR, whereas of 47 patients with a BMA14th15%, 38 (81%) were NR (p = 0.0001). The test sensitivity and specificity were 66.7% and 80.9%, respectively.Our data suggest that BMA14th may be a predictive test for CR, helping to identify NR patients early in their disease. Further studies are needed to establish the practical implications of the results of our study.

The expression of CXCR4 (CD184) has been associated with poor prognosis in Acute Myeloid Leukemia (AML) and it has also been suggested that the CXCL12(SDF-1a)/CXCR4 interaction contributes to the resistance of leukemia cells to chemotherapy-induced apoptosis. Inhibition of CXCR4 was found to enhance chemotherapy-induced apoptosis in a subset of leukemic myeloblasts that carry Flt3 mutations and to overcome chemoresistance associated with stromal activity. NPM variants with a cytoplasmic localization represent the most common mutation detected in myeloid malignancies and are associated with a favourable clinical outcome. A recent study provides biological evidence for a novel role for NPM as a negative regulator of CXCR4 signalling induced by CXCL12: suppression of NPM expression enhanced chemotactic responses to CXCL12, and conversely, over-expression of a cytosolic NPM mutant reduced chemotaxis induced by CXCL12. We investigated whether CD184 expression is a negative predictor factor for response to chemotherapy and if there is clinical evidence that NPM mutations could overcome chemoresistance to induction therapy in this subset of patients. The expression of CD184 was analyzed by flow cytometric methods in a group of 70 cases of adult AML at onset of disease, diagnosed at our Institution since January 2006. The diagnosis was performed according to FAB/WHO criteria; all patients received intensive chemotherapy according to institutional protocols. There were 34 males and 36 females and median age was 46 years (range 18–65). AML cells were gated based upon their CD45 expression and samples were considered positive if CD184 was expressed by more than 20% of blasts. CD184 was positive in 45 and negative in 25 cases. There was no significant difference between the two groups in terms of sex, age, Hb level, WBC and Plt counts, percentage of blasts, and occurrence of the NPM mutation. The CR rate was 45% in CD184+ and 82% in CD184- (p=0.03); among CD184+ cases, the CR rate was significantly higher in NPMc+ cases, (p=0.03). Our results show that CD184 expression is associated with a lower rate of CR after induction therapy and this association is stronger in NPM unmutated cases, suggesting that CD184 expression is a negative predictive factor for response to chemotherapy. Further data are needed to verify if the biological role of the cytosolic NPM mutant as a negative regulator of CXCR4 signalling induced by CXCL12 could have a clinical role contributing to overcome the resistance of leukemic cells to induction chemotherapy.

// Francesco Albano 1 , Antonella Zagaria 1 , Luisa Anelli 1 , Paola Orsini 1 , Crescenzio Francesco Minervini 1 , Luciana Impera 1 , Paola Casieri 1 , Nicoletta Coccaro 1 , Giuseppina Tota 1 , Claudia Brunetti 1 , Angela Minervini 1 , Domenico Pastore 1 , Paola Carluccio 1 , Anna Mestice 1 , Angelo Cellamare 1 , and Giorgina Specchia 1 1 Department of Emergency and Organ Transplantation (D.E.T.O.), Hematology Section, University of Bari, Bari, Italy. Correspondence: Francesco Albano, email: // Keywords : acute promyelocytic leukemia, LEF1; prognosis; outcome. Received : November 20, 2013 Accepted : December 19, 2013 Published : December 19, 2013 Abstract Lymphoid enhancer-binding factor 1 ( LEF1 ) is a downstream effector of the Wnt/ β-catenin signaling pathway. High LEF1 expression has been reported as a prognostic marker in hematologic malignancies. We evaluated the prognostic significance of LEF1 expression in 78 adult acute promyelocytic leukemia (APL) patients. APL samples were dichotomized at the median value and divided into: LEF1 low and LEF1 high . LEF1 high patients had lower WBC counts at baseline and were less likely to carry a FLT3 -ITD than LEF1 low patients. Early death occurred only in the LEF1 low group. Moreover, LEF1 low expression was associated with a high Sanz score. Survival analysis of 61 APL patients < 60 years revealed that the LEF1 high group had a significantly longer overall survival (OS). Cox analysis for OS confirmed only LEF1 expression as an independent prognostic factor. Of the 17 patients over the age of 60, those in the LEF1 high group showed a higher median survival. In silico analysis identified 9 differentially expressed, up-modulated genes associated with a high expression of LEF1 ; the majority of these genes is involved in the regulation of apoptosis. Our study provides evidence that LEF1 expression is an independent prognostic factor in APL, and could be used in patients risk stratification.

An experimental test campaign has been performed by means of Pressure Sensitive Paint Technique (PSP) and Particle Velocimetry Anemometry (PIV) on a laminar airfoil at low Reynolds number. Experimental investigation has been intended to get detailed information about pressure and velocity fields and to detect possible laminar-separation bubble location on the concave region of the airfoil. Pressure distributions around the model have been used as “starting point” to individuate possible separation locations on the airfoil and to better direct macro-PIV investigations inside the airfoil boundary layer. Results will be presented in terms of pressure coefficient chord-wise distribution and velocity profiles on the model surface. Velocity profiles have been processed to get boundary layer integral parameters (displacement and momentum thickness). Moreover, pressure and velocity fields have been integrated to obtain the airfoil lift and drag coefficient in the investigated range of angles of attack.

Smoldering multiple myeloma (SMM), an asymptomatic plasma cell neoplasm, is currently diagnosed according to the updated IMWG criteria, which reflect an intermediate tumor mass between monoclonal gammopathy of undetermined significance (MGUS) and active MM. However, SMM is a heterogeneous entity and individual case may go from an “MGUS-like” behavior to “early MM” with rapid transformation into symptomatic disease. This wide range of clinical outcomes poses challenges for prognostication and management of individual patients. However, initial studies showed a benefit in terms of progression or even survival for early treatment of high-risk SMM patients. While outside of clinical trials the conventional approach to SMM generally remains that of close observation, these studies raised the question of whether early treatment should be offered in high-risk patients, prompting evaluation of several different therapeutic approaches with different goals. While delay of progression to MM with a non-toxic treatment is clearly achievable by early treatment, a convincing survival benefit still needs to be proven by independent studies. Furthermore, if SMM is to be considered less biologically complex than MM, early treatment may offer the chance of cure that is currently not within reach of any active MM treatment. In this paper, we present updated results of completed or ongoing clinical trials in SMM treatment, highlighting areas of uncertainty and critical issues that will need to be addressed in the near future before the “watch and wait” paradigm in SMM is abandoned in favor of early treatment.