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The translocation t(5;12)(q31;p13)/ETV6::ACSL6 is a rare cytogenetic abnormality, although it is reported in various myeloid malignancies. To date, only 16 cases of t(5;12) and ETV6::ACSL6 rearrangement, confirmed by either molecular or Fluorescence In Situ Hyridization (FISH) analysis, have been reported. Eosinophilia is a distinctive and common feature associated with this rearrangement. Although few cases have been described, the prognosis of patients with ETV6::ACSL6 is considered poor. We report two additional cases of t(5;12)(q31;p13)/ETV6::ACLS6 rearrangement and eosinophilia. Unusually, in our cases, the ETV6::ACSL6 rearrangement occurred at the relapse of Acute Myeloid Leukemia (AML) patients who had t(6;9)(p23;q34)/DEK::NUP214 rearrangement at disease onset. The concurrence of these two rare abnormalities has never been reported and may suggest a cooperative role of t(5;12) and t(6;9), leading to disease relapse. Moreover, at relapse, both cases presented with eosinophilia, further strengthening the association of t(5;12) with eosinophilia in myeloid malignancies. Given the poor prognosis and the non-responsiveness to tyrosine kinase inhibitors of cases of ETV6::ACSL6 rearrangement, in contrast to cases of ETV6::PDGFRB rearrangement, we recommend the introduction of testing for this abnormality in myeloid malignancies with eosinophilia.

Chromosomal rearrangements involving 3q26 are recurrent findings in myeloid malignancies leading to MECOM overexpression, which has been associated with a very poor prognosis. Other 3q abnormalities have been reported and cryptic MECOM rearrangements have been identified in some cases. By fluorescence in situ hybridization (FISH) analysis, we investigated 97 acute myeloid leukemia/myelodysplastic syndrome patients with various 3q abnormalities to determine the role and the frequency of the involvement of MECOM. We identified MECOM rearrangements in 51 patients, most of them showed 3q26 involvement by chromosome banding analysis (CBA): inv(3)/t(3;3) (n = 26) and other balanced 3q26 translocations (t(3q26)) (n = 15); the remaining cases (n = 10) showed various 3q abnormalities: five with balanced translocations involving 3q21 or 3q25; two with homogenously staining region (hsr) on 3q; and three with other various 3q abnormalities. Complex rearrangements with multiple breakpoints on 3q, masking 3q26 involvement, were identified in cases with 3q21/3q25 translocations. Furthermore, multiple breaks were observed in two cases with t(3q26), suggesting that complex rearrangement may also occur in apparently simple t(3q26). Intrachromosomal gene amplification was another mechanism leading to MECOM overexpression in two cases with hsr on 3q. In the last three cases, FISH analysis revealed 3q26 involvement that was missed by CBA because of metaphases' suboptimal quality. All cases with MECOM rearrangements showed overexpression by real-time quantitative PCR. Finally, MECOM rearrangements can occur in patients with 3q abnormalities even in the absence of specific 3q26 involvement, underlining that their frequency is underestimated. As MECOM rearrangement has been associated with very poor prognosis, its screening should be performed in patients with any 3q abnormalities.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterised by a specific chromosomal translocation, t(9;22)(q34;q11), resulting in the Philadelphia (Ph) chromosome. T...

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1.

// Simona Luatti 1 , Carmen Baldazzi 1 , Giulia Marzocchi 1 , Gaia Ameli 1 , Maria Teresa Bochicchio 1 , Simona Soverini 1 , Fausto Castagnetti 1 , Mario Tiribelli 2 , Gabriele Gugliotta 1 , Giovanni Martinelli 1 , Michele Baccarani 1 , Michele Cavo 1 , Gianantonio Rosti 1 , Nicoletta Testoni 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. and A. Seragnoli”, University of Bologna, “S Orsola-Malpighi” University Hospital, Bologna, Italy 2 Division of Hematology and Bone Marrow Transplantation, Azienda Ospedaliero-Universitaria di Udine, Udine, Italy Correspondence to: Simona Luatti, email: simonaluatti@gmail.com Keywords: CML, BCR/ABL, Philadelphia chromosome, FISH, TKI Received: September 23, 2016 Accepted: January 16, 2017 Published: February 16, 2017 ABSTRACT At diagnosis, about 5% of Chronic Myeloid Leukemia (CML) patients lacks Philadelphia chromosome (Ph), despite the presence of the BCR/ABL rearrangement. Two mechanisms have been proposed about the occurrence of this rearrangement: the first one is a cryptic insertion between chromosomes 9 and 22; the second one involves two sequential translocations: a classic t(9;22) followed by a reverse translocation, which reconstitutes the normal morphology of the partner chromosomes. Out of 398 newly diagnosed CML patients, we selected 12 Ph-negative cases. Six Ph-negative patients treated with tyrosine kinase inhibitors (TKIs) were characterized, in order to study the mechanisms leading to the rearrangement and the eventual correlation with prognosis in treatment with TKIs. FISH analysis revealed cryptic insertion in 5 patients and classic translocation in the last one. In more detail, we observed 4 different patterns of rearrangement, suggesting high genetic heterogeneity of these patients. In our cases, the BCR/ABL rearrangement mapped more frequently on 9q34 region than on 22q11 region, in contrast to previous reports. Four patients, with low Sokal risk, achieved Complete Cytogenetic Response and/or Major Molecular Response after TKIs therapy. Therapy resistance was observed in one patient with duplication of BCR/ABL rearrangement and in another one with high risk. Even if the number patient is inevitably low, we can confirm that the rare Ph-negative CML patients do not constitute a “warning” category, meanwhile the presence of further cytogenetic abnormalities remains an adverse prognostic factor even in TKI era.

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1. Overall, we have demonstrated that in MF different mutations lead to phenotypic and functional alterations in different immune subsets that may have a potential role in disease progression and susceptibility to infections.

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease.

e46 therapies. Activating mutations of KRAS, NRAS and BRAF have been reported in approximately 23%, 20% and 6% of MM cases. Detection of these actionable mutations currently requires bone marrow (BM) aspirates that are invasive and can yield suboptimal samples. Targeted, ultra-deep sequencing of cfDNA is a promising tool for accessing the tumour genome that has not been studied in MM.We set out to identify actionablemutations andmutational load through cfDNA analysis and compare the results to matched tumourderived DNA.Methods: MM patients (pts) with active disease were identified and consented to have their peripheral blood drawn for analysis. Where possible, matched tumour DNA derived from BM CD138+ selected cells was also obtained and sequenced. cfDNA was extracted from 3-15 mL of plasma using the QIAamp Circulating Nucleic Acid Kit and tagged with sequencing barcodes for subsequent pooling. All exons of KRAS, NRAS, BRAF, PIK3CA and EGFR genes were isolated using a custom hybrid capture panel (IDT xGen Lockdown) and sequenced on an Illumina HiSeq 2000. Reads were aligned to the human genome reference (hg19) using bwa and somatic mutations were detected using muTect. Results: We have collected 39 plasma samples from 35 pts, 10 newly diagnosed and 25 from relapsed pts who had a median of 3 prior lines of therapy (range 1-7). The median yield of cfDNA was 17.4ng/mL of plasma (range: 2.7298) with a trend towards higher yield from pts with ISS stage 3 disease. Somatic mutations in at least 1 of the 5 genes were present in 22 of 35 pts (63%) with mutant allele frequencies ranging from 0.1942%.Mutations in KRAS, NRAS and/or BRAF were identified in 21 pts with some having multiple actionable mutations (Figure 1). Twenty-five samples from 23 pts had matching BM-derived tumour DNA from which 26 coding mutations were detected and all but two were found in the cfDNA (92.3% sensitivity). In addition, 6 mutations (4 BRAF in one patient) were only detected in cfDNA, making for a specificity of 97.1%. Conclusion: Ultra-deep targeted sequencing of cfDNA derived from blood of MM pts is feasible and able to identify tumour-derived mutations with high sensitivity and specificity. cfDNA analysis identified key actionable mutations and thus represents a non-invasive source of tumour DNA that can potentially inform treatment options for pts with MM.

BACKGROUND: The incidence of chronic myeloid leukemia (CML) increases with age, but it is unclear how the characteristics of the disease vary with age. In children, where CML is very rare, it presents with more aggressive features, including huge splenomegaly, higher cell count and higher blast cell percentage. PATIENTS AND METHODS: To investigate if after childhood the disease maintains or loses these characteristics of aggressiveness, we analyzed 2784 adult patients, at least 18 years old, registered by GIMEMA CML WP over a 40-year period. RESULTS: Young adults (YAs: 18-29 years old) significantly differed from adults (30-59 years old) and elderly patients (at least 60 years old) particularly for the frequency of splenomegaly (71%, 63% and 55%, P < 0.001), and the greater spleen size (median value: 4.5, 3.0 and 1.0 cm, P < 0.001). According to the EUTOS score, that is age-independent, high-risk patients were more frequent among YAs, than among adult and elderly patients (18%, 9% and 6%, P < 0.001). In tyrosine kinase inhibitors-treated patients, the rates of complete cytogenetic and major molecular response were lower in YAs, and the probability of transformation was higher (16%, 5% and 7%, P = 0.011). CONCLUSIONS: The characteristics of CML or the host response to leukemia differ with age. The knowledge of these differences and of their causes may help to refine the treatment and to improve the outcome. CLINICAL TRIAL NUMBERS: NCT00510926, NCT00514488, NCT00769327, NCT00481052. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Introduction: Myelofibrosis (MF), a clonal disorder of the hemopoietic stem/progenitor cell, is characterized by distinctive abnormalities in megakaryocyte (MK) development. A proliferation of dysplastic MK with disturbance of nuclear/cytoplasmic maturation is observed. In addition to driver mutations in Janus Kinase 2 (JAK2) and Calreticulin (CALR) genes, chronic inflammation has emerged as a key-player in MF pathogenesis. Here, we analyzed the potential contribution of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α and Tissue Inhibitor of Metalloproteinases (TIMP)-1 to MF dysmegakaryocytopoiesis. Methods: We studied 10 patients with MF at diagnosis while 6 patients had received previous treatment for MF. In all cases, therapies had been discontinued for at least two months before peripheral blood samples collection. To characterize the mutational status, JAK2V617F and CALR exon 9 mutations were assessed by quantitative PCR-based allelic discrimination assay and Next Generation Sequencing approach, respectively. To quantify MK progenitors, circulating CD34+CD41+ cells of MF patients (10 JAK2V617F and 6 CALR mutated) and controls (cord blood (CB); 8 cases) were identified by flow cytometry analysis. To evaluate the pro-inflammatory profile, IL-1β, TNF-α and TIMP-1 were measured in plasma of patients (10 JAK2V617F and 6 CALR mutated) and healthy subjects (10 cases) by ELISA. To determine whether and to what extent crucial factors of the inflammatory microenvironment may affect the in vitro growth of MK progenitors, Colony Forming Units-Megakaryocyte (CFU-MK) growth of circulating CD34+ cells from MF patients (6 JAK2V617F and 6 CALR mutated) and CB (6 cases) was assessed with a collagen-based medium in the presence or absence of TIMP-1, TNF-α and IL-1β (alone or in combination). Pure and mixed CFU-MK were quantified on the basis of CD41 immunostaining. Results: We found that, irrespective of mutational status, IL-1β, TNF-α and TIMP-1 plasma levels were significantly increased in MF patients. The frequency of circulating MF-derived CD34+CD41+ cells was significantly higher than the CB counterparts. However, a significantly higher proportion of cells was associated with CALR compared to JAK2V617F mutated patients. The growth of pure CFU-MK from untreated cells of the JAK2V617F mutated patients was significantly decreased as compared to that of CALR mutated patients and the CB counterparts. Factors alone did not stimulate the CFU-MK growth from CB CD34+ cells. Conversely, IL-1β had stimulatory activity on pure MK colony formation of JAK2V617F, but not CALR, mutated patients and the growth of MK progenitors from MF patients was significantly inhibited by TNF-α, irrespective of mutational status. TIMP-1 was ineffective. Factors in combination did not significantly modify the growth of pure CFU-MK from patients/CB as compared with factors alone. The growth of mixed MK colonies from untreated cells was absent (JAK2V617F mutated patients) or very low (CALR mutated patients and CB). Only TNF-α (alone or in combination (IL-1β+TNF-α and IL-1β+TNF-α+TIMP-1)) increases the growth of mixed MK colonies of patients harboring CALR mutation and CB. Interestingly, factors alone or in combination failed to stimulate the growth of mixed MK colonies from JAK2V617F mutated patients. Conclusions: Taken together these results demonstrate that in MF signals provided by the inflammatory microenvironment promote a mutation-associated functional dysregulation of MK progenitors and suggest a mechanism by which chronic inflammation may play a role in the development/maintenance of the abnormal megakaryocytopoiesis. Disclosures Martinelli: BMS: Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau. Cavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria.

We investigated whether low-level Bcr–Abl kinase domain mutations can be detected in patients who have stable responses to first-line nilotinib–like it is known to happen in patients receiving imatinib. We screened for mutations twelve such patients by cloning and sequencing. Only one case was found to harbor mutations at low levels (including a T315I). However, major molecular response (MMR) was maintained and it even improved to complete molecular response. Our results suggest that a) Bcr–Abl mutations, even at low level, seem to be very rare in patients in MMR on first-line nilotinib; b) low-level mutations do not always predict for subsequent relapse.

Clonal cytogenetic abnormalities (CAs) in Philadelphia negative (Ph−) metaphases have been widely observed during imatinib treatment in patients with chronic myeloid leukemia (CML) [1,2]. Recently, the appearance of such abnormalities in some CML patients, treated with dasatinib after imatinib failure, has been described [3,4]. To the best of our knowledge, only one patient has been reported to develop a cytogenetic abnormality in Ph− clone during nilotinib treatment, following imatinib [5]. The incidence and consequences of CAs in Ph− cells during treatment with second generation tyrosine kinase inhibitors (TKIs) should be extensively investigated; this issue, deserves a particular attention, since these new drugs put a far higher pressure on the leukemic and the residual normal hematopoiesis than imatinib.

Abstract 2196 Poster Board II-173 BACKGROUND: Imatinib (IM) 400 mg daily is the standard treatment for Chronic Myeloid Leukemia (CML) in early chronic phase (ECP). The European LeukemiaNet (ELN) recommendations were designed to help identify ECP CML patients responding poorly to front-line IM, suggesting, at given time points, when the treatment strategy should be changed (”failure”), or when “the long-term outcome of the treatment would not likely be as favourable” (“suboptimal response”). Suboptimal response is a “grey zone”: the patient may still have substantial benefit from continuing IM, but other therapies should be considered. AIM: To assess the outcome of “failure” and “suboptimal responders” Philadelphia-positive (Ph+) CML patients in a large multicentric, nationwide experience. METHODS: Between January 2004 and April 2007, 559 patients were enrolled in an observational study and in 2 independent intervention studies of the GIMEMA CML WP (Clin Trials Gov. NCT00514488 and NCT00510926). Response monitoring was based on conventional cytogenetic examination of bone marrow cell metaphases every 6 months and RT Q-PCR evaluations of blood cells after 3, 6, 12 months, and every 6 months thereafter. Definitions: major molecular response (MMR): BCR-ABL/ABL ratio < 0,1%IS; failure (according to ELN criteria): no hematologic response (HR) at 3 months, no complete HR (CHR) at 6 months, no cytogenetic response (CgR) at 6 months, no partial CgR (PCgR) at 1 year, no complete CgR (CCgR) at 18 months, loss CHR or CCgR, progression or death; suboptimal response (according to ELN criteria): no CHR at 3 months, no PCgR at 6 months, no CCgR at 12 months, no MMR at 18 months ; optimal response: non-suboptimal and non-failure at each time-point; event: failure or treatment discontinuation for any reason. All the calculations have been made according to the intention-to-treat principle. RESULTS: The patients who fitted the ELN criteria for failure had a significantly lower probability of subsequently achieving a CCgR and a MMR, and had a significantly lower overall survival (OS), failure-free survival (FFS) and event-free survival (EFS). The patients who fitted the ELN definitions of suboptimal response at 6 months (data not shown) and at 12 months (figure 1) had a significantly lower probability than “optimal” responders of subsequently achieving a CCgR and a MMR, and a significantly poorer FFS and EFS (figure 1), while the OS was not different in the two groups (90% and 95%, p= 0.35). CONCLUSIONS Our data confirms that suboptimal responders at 6 and at 12 months have a poorer outcome with respect to “optimal” responders, comparable to the outcome of failure patients. Acknowledgments: European LeukemiaNet, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Abstract 2205 Poster Board II-182 Nilotinib is an effective and registered treatment of chronic myeloid leukemia (CML) after imatinib failure. Its efficacy as frontline treatment has been explored in phase 2 trials from MDACC and Italian GIMEMA , whose results have been presented recently (Cortes ASH Rosti, EHA). Here we present a detailed analysis of the safety profile of nilotinib 800 mg daily in the CML early chronic phase (ECP) setting. Briefly, 73 ECP patients (median age 51 yrs, range 18-83 yrs, 21/73 – 29% - ≥ 65 yrs at enrolment) received nilotinib at a dose of 400 mg BID. With a median follow-up of 15 months (range 12-24 months), the CCgR rate at 1 yr was 96%, and the major molecular response (MMolR) rate 85%. During the first 365 days, the treatment was interrupted at least once in 38 patients (52%; overall, 86 interruptions), with a median cumulative duration of drug interruption of 19 days (5.2% of 365 days) per patient (range 3-169 days); 35 pts (48%) received the full prescribed dose. The proportion of patients with ≥ 1 interruption decreased during the first and second quarter and second half (37%, 25% and 22% respectively). The mean daily dose was 600-800 mg, 400-599 mg, and less than 400 mg in 74%, 18% and 8% of patients, respectively. Four AEs (≥ grade 2) accounted for the great majority of dose interruptions: bilirubin increase (38%, no gr. 4), skin rash and/or pruritus (37%, no gr. 4), asymptomatic amylase and/or lipase increase (16%, gr. 4: 4%) (no pancreatitis), transaminases increase (19%, no gr. 4). Notably, only 3 events of peripheral edema/fluid retention have been recorded so far (2 gr 1, 1 gr. 2). No pleural or pericardial effusion. Only one pt permanently discontinued nilotinib for recurrent amylase and lipase increase gr. 3-4 after 7 months on nilotinib, without pancreatitis (normal ECO scan and MRI): the pt. is on imatinib 400 mg daily from 12 months, maintaining the CCyR but loosing MMolR on imatinib. The transient hyperglicemia (gr. 2 and 3: 6%) did not lead to any treatment discontinuation. The hematopoietic toxicity (grade 3-4) was negligible: only 5 events (3 neutropenias and 2 thrombocytopenias) in 5 pts (7%) (all within 3 months from treatment start: 431/438 q2weeks scheduled blood counts evaluable). Nilotinib 800 mg daily is feasible, safe and very effective in ECP CML (ClinicalTrials Gov.NCT00481052). ACKNOWLEDGEMENTS: The Italian Association Against Leukemia-lymphoma and myeloma (BolognAIL), The Fondazione del Monte di Bologna e Ravenna, The Italian Ministery of Education (PRIN 2005, No. 20050 63732_003, and PRIN 2007, No 2007F7 AE7B_002), The University of Bologna, The European Union (European LeukemiaNet). Disclosures: No relevant conflicts of interest to declare.

Background Ponatinib, a potent third generation pan BCR-ABL inhibitor, has recently shown relevant activity against native and mutant forms of BCR-ABL, including the TKI resistant T315I mutant. The aim of this compassionate protocol was to confirm and evaluate the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML. Design and Methods Ponatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F: 8/9) have been treated with Ponatinib (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL (10 p190, 4 p210) and 3 blast phases of CML (2 Myeloid and 1 Lymphoid, p210). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 754 days (range 46-2264). All the patients were resistant or intolerant to previous TKIs (median number of previous TKIs: 2; range 1-3). Standard chemotherapy was previously performed in 7/17 patients (41%). Four patients (23%) had previously received allogeneic stem cell transplantation. At the time of enrolment, median Hb, PLTs and WBC values were 10,9 g/dl (range 8.6-13.9), 139000/mmc (range 14000-325000) and 4300/mmc (range 1700-17000), respectively. In 6 out of 17 patients, additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (9 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). No mutations were detected in 4 patients. Results The median treatment duration was 139 days (range 14-540+). Causes of treatment stop were: progression disease (5 patients), savage allogenic stem cell transplantation (6 patients), drug intolerance (1 patient), consisting in grade III headache. With a median follow up of 284 days (range 8-540+), a maHR was obtained in 13/17 patients (76%). After one month of treatment, a reduction of BCR-ABL fusion transcript level was observed in 9/15 patients (60%). For two patients the follow up is too short to be evaluable. The level became undetectable in 4 patients (3 presenting with T315I mutation). After treatment, T315I mutation disappeared in 6 out of the 9 patients who showed this molecular alteration at the beginning of therapy. At the time of this report, 6/17 patients are still on study (35%). Five patients died due to progression disease. As expected, the drug was well tolerated. Non-hematologic adverse events were described in 7/17 patients (grade >III skin rash in 3 patients; grade>II serum lipase increase in 2 patients; grade>II myalgia in 1 patient; grade III headache). Conclusion In our experience, the activity of Ponatinib in advanced Ph+ leukemias, mainly in T315I mutated patients, was confirmed. No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients early on who could take advantage of this treatment. Acknowledgments Work supported by European LeukemiaNet, AIRC, PRIN 2010-2011, University of Bologna and BolognAIL. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic alterations that provided important prognostic information capable to guide therapy and predict outcome. The reported TP53 mutation rate in AML is low (2.1%). By contrast, the incidence of FLT3 disruption is frequent (20-30%) and is an independent predictor of unfavourable outcome of acquired clinical resistance to FLT3 inhibitors. TP53 mutations in AML with a complex karyotype (CK) is higher (69-78%), but few data are available for association between the most common AML associated lesions such as FLT3, NPM1, DNMT3A, IDH2 and TP53 mutation or CK. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the molecular abnormalities, the correlation with known molecular alterations (FLT3, NPM, etc.) and the prognostic role TP53 mutations in adult AML pts. Patients and Methods: 886 AML patients were analysed for cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, DNMT3A, IDH1-2 etc). Of these, 200 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq 2000 platform (38/200) and were correlated with cytogenetic analysis. Results: 55 pts (27.5%) showed 3 or more chromosome abnormalities (CK-AML), 83 (41.5%) presented one or two cytogenetic abnormalities (other-AML) and 43 pts (21.5%) have normal karyotype (nK). In 19 cases the karyotype was not available. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations (32 deleterious point mutations; 4 deletions); seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had CK while only 6/29 (21%) mutated pts have “no CK”. Overall, between pts with CK, TP53 frequency is 41.8% (P>0,0001). 120 pts were analysed for concomitant presence of TP53 mutations and FLT3/NPM1 disruption/mutation: revealing a significant relation between pts with FLT3-ITD or NPM1 and TP53 wild-type (p = 0.043 and 0.022 respectively). As for clinical outcome alterations of TP53 were significantly associated with poor outcome (OS and EFS p WES analysis done in 38 pts (33 TP53 wt and 5 pts TP53 mutated) revealed no genes exclusively mutated in the 5 TP53 mutated pts. Conclusions: Our data demonstrated that TP53 mutations occur in 14.5% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Cristina Papayannidis, Elisa Zuffa, Carmen Baldazzi, Antonella Padella, Eugenia Franchini, Ilaria Iacobucci, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Francesca Volpato, Federica Cattina, Giorgia Simonetti, Maria Chiara Fontana, Maria Teresa Bochicchio, Federica Frabetti, Elisa Lani, Katia Mancuso, Beatrice Zannetti, Simona Luatti, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. TP53 mutations are mutually exclusive with FLT3 and NPM mutations in AML patients and are strongly associated with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2015-4906

Molecular and cytogenetic characterization of a new case of t(5;17)(q35;q21) variant acute promyelocytic leukemia

Background: Ponatinib, a third generation BCR-ABL inhibitor, has shown relevant activity against native and mutant forms of BCR-ABL, including the T315I mutant. The aim of this compassionate protocol was to confirm the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML. Design and Methods: Ponatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F=8/9) have been treated (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL and 3 blast phases of CML (11 p190, 5 p210, 1 p230). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 2 years (range 0.2-6). Five patients (29%) had previously received an allogenic stem cell transplantation. All the patients were resistant or intolerant to previous TKIs (11 patients received >=2 TKIs). Median Hb, PLTs and WBC values were 10,1 g/dl (range 8.3-13.9), 100000/mmc (range 14000-325000) and 4400/mmc (range 1700-17000), respectively. In 6 out of 17 patients (35%), additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (8 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). Results: The median treatment duration was 153 days (range 42-594+). With a median follow up of 8 months (range 2-21+), a maHR was obtained in 12/17 patients (71%). Among these, 6 were T315I mutated. After one month of treatment, a reduction of BCR ABL fusion transcript to undetectable level was observed in 3/17 patients (18%), which increased to 29% (5/17) after two additional cycles. The progression free survival (PFS) at 12 months was 35% (median 57 days), without differences between patients with T315I mutations and patients harboring other mutations. Furthermore, we observed that, in responder patients, mutations disappeared in all except one case. In contrast, in resistant patients, a specular mutational profile before and after treatment was detected. Currently, 6/17 patients are still on study (35%). Six patients died due to progression disease. In five cases, the response allowed the patients to proceed to a savage allogenic stem cell transplantation. The drug was well tolerated. No SAEs were detected and no thrombotic arterial/venous events occurred. Conclusion: In our experience, the activity of Ponatinib in advanced Ph+ leukemias was confirmed.No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients who could take advantage of this treatment. Acknowledgments: Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Caterina De Benedittis, Simona Soverini, Ilaria Iacobucci, Maria Chiara Abbenante, Chiara Sartor, Maria Teresa Bochicchio, Anna Ferrari, Claudia Venturi, Valentina Robustelli, Andrea Ghelli Luserna di Rorà, Viviana Guadagnuolo, Emanuela Ottaviani, Nicoletta Testoni, Carmen Baldazzi, Simona Luatti, Sarah Parisi, Stefania Paolini, Alberto Conficoni, Federica Frabetti, Elisa Lani, Silvia Piccari, Paolo Di Bartolomeo, Roberto Di Lorenzo, Renato Fanin, Giuseppe Cimino, Fabio Ciceri, Giovanni Martinelli. Ponatinib is well tolerated and active in patients with relapsed/refractory philadelphia positive leukemias: The Bologna experience. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT312. doi:10.1158/1538-7445.AM2014-CT312

The transcriptional factor nuclear factor-erythroid 2 (NF-E2) is one of the few transcription factors known to be functionally linked to the megakaryocytic lineage, where it regulates terminal megakaryocyte maturation and platelet formation. However, the regulation of NF-E2 expression in megakaryocytic cells has not been extensively evaluated. In particular, no data have been reported on the effect of negative regulators of megakaryocytopoiesis on NF-E2 expression. This study investigated the in vitro effects of two negative regulators of megakaryocytopoiesis, such as interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) on the expression of NF-E2 transcription factor in megakaryoblastic cell lines (Hel and MK1) and in normal CD34-derived megakaryocytic cells. For this purpose, we used quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA NF-E2 isoforms (a and f) and flow-cytometry analysis to evaluate NF-E2 protein expression. Our results demonstrated that TGF-beta1 did not inhibit NF-E2 mRNA and protein expression of either maturating or fully mature normal megakaryocytic cells as well as that of the two cell lines. By contrast, IL-4 downmodulates the expression of NF-E2 transcription factor at both mRNA and protein levels in normal maturating megakaryocytic cells and in the megakaryoblastic cell lines. NF-E2 expression of normal mature megakaryocytes was not affected by IL-4. Thus, the results of the present investigation demonstrate that NF-E2 transcription factor is involved not only in terminal megakaryocyte maturation but also in the negative regulation of the early phase of megakaryocyte development.

Thrombocytopoiesis of 21 multiple myeloma patients undergoing single or double transplant regimen was characterized by measuring the level of reticulated platelets and plasma glycocalicin. Since reticulated platelets are an index of thrombopoietic activity and glycocalicin plasma values are related to platelet damage and turnover, it may be possible to perform a novel type of analysis of the thrombopoietic compartment during the mobilizing regimen and during transplant-related chemotherapy. Patients underwent mobilizing therapy and first transplant. Some randomized patients also underwent a second transplant with mobilized peripheral blood stem cells. The results show that the percentage of reticulated platelets decreased after therapy and then gradually increased in the recovery phase either during first or second transplant. By contrast, the percentage of reticulated platelets increased until day +8 and then gradually decreased during the mobilizing regimen. The glycocalicin index (glycocalicin plasma value normalized for the individual platelet count) increased significantly both during the course of mobilization and after transplant-related chemotherapy when the platelet number was at its nadir. However, the glycocalicin index was more elevated after transplant-related chemotherapy than after the mobilizing regimen. Our findings suggest that chemotherapy-related thrombocytopenia may be due to a dual mechanism: thrombocytopenia results from decreased platelet production in addition to increased platelet damage and possible destruction.

No abstract available.

Abstract 2174 Poster Board II-151 Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disease which typically presents in chronic phase (CP), whose malignant progenitor cells proliferate rapidly, still retaining their ability to differentiate. If left untreated the disease can rapidly progress to accelerated phase and blast crisis. Although the treatment has been dramatically improved with introduction of Glivec therapy, the use of the Sokal and the Euro prognostic scores has remained an essential clinical tool to stratify CML patients at diagnosis based on different evolutive risk and to guide treatment decisions. To further optimize the management of the disease it is critical to gain a better understanding of regulatory pathways involved in the intrinsic heterogeneity of CML and propensity to progress. To that end our effort has been focused on identifying a molecular signature associated with a risk of the disease progression. Here we present data obtained from our study of gene expression profiles (GEP) aimed at identifying genes and pathways which could predict the disease course of CP-CML patients at the onset of the disease. The study was performed on highly enriched CD34+ cells from peripheral blood obtained from patients with untreated CML in CP. GEP was performed by using the Affymetrix HG-U133 Plus 2.0 platform. Raw data were normalized by using the RMA algorithm and filtered. Genes associated with Sokal risk score were selected by a moderate t-statistic (Limma package, p-value threshold = 0.01). Hierarchical clustering was performed with TIGR MeV. Overall, 34 pts were included in the present analysis. In the initial part of the study, the first 20 pts (the “training set”) were successfully assayed for global GEP and microarray data and were used to define a set of genes differentially expressed in high (H) (7 pts) vs. low (L) (13 pts) Sokal risk pts. We identified 84 probes sets and the clustering of their GEP showed an homogeneous pattern in H Sokal risk pts, where the most significantly involved process networks (as defined by GeneGo software) were: “Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity” (PLCB1, CALM1, PRKCA, PPPIR14A, MYL4), “Cytoskeleton remodeling_TGF and WNT” (ACTN1, CFL2, TCF7L2) and “Development_WNT signaling pathway” (WNT6, FZD3, TCF4, VEGF-A). Of interest, among the most significantly up-regulated genes, we identified PVT1, a non-coding gene located on chromosome 8, close to c-Myc gene, which encodes for several miRNA able to activate c-Myc gene transcription. Among the most significantly down-regulated genes is PLCB1, which we have recently described as being deleted in myelodysplastic syndromes and in acute myeloid leukemia. In the second part of the study, the 84 probes set were tested on an independent test set of 14 pts, including 4 H, and 10 L Sokal risk pts., thus showing that the GEP clustering displayed the same feature which we have observed in the training set. In conclusion, our study has identified a distinct array of genes at diagnosis which might be involved in driving the evolutive risk of CML and potentially, this approach could be of value to better define patients who may need an optimized treatment. Supported by:Novartis Oncology, TOPS Correlative Studies, PRIN, AIRC, AIL, FIRB 2006, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare.

Dasatinib (BMS-354825) is a second-generation BCR-ABL inhibitor active against several imatinib-insensitive BCR-ABL mutant forms. We have treated in the phase II program with dasatinib a total of forty-five Ph+ pts who were resistant to or intolerant of imatinib. At the time of writing, twenty pts have failed to respond to or relapsed on dasatinib therapy. In order to assess which pre-existent or emerging ABL kinase domain (KD) mutations are challenging for dasatinib clinical efficacy, we retrospectively analyzed ABL KD sequences before the start of treatment and every month thereafter, until dasatinib discontinuation. Mutation monitoring was done by D-HPLC, followed by sequencing in D-HPLC-positive cases. Eight pts had primary resistance to dasatinib (Table 1). In all cases, a T315I or a F317L mutation was already detectable before the onset of treatment or became detectable after one month. The mutations persisted up to the time of disease progression, which occurred at a median of 1.5 months (range, 1–4) from dasatinib start. Twelve pts had acquired resistance to dasatinib (Table 1). Relapse occurred after a median of 7.5 months (range, 3–15) from dasatinib start. Mutation analysis performed before the onset of treatment showed that five of these pts had a wild-type ABL sequence, while the remaining seven pts had evidence of various imatinib-resistant KD mutations (G250E, Y253H, E255K, D276G, M351T). At the time of relapse, however, most of the original mutant clones had disappeared, whereas mutations at residues 315 (T315I or T315A) and/or 317 (F317L or F317I) had invariably emerged in all but one pt. This pt was found to have developed a novel K356R mutation which is now under characterization. Our results indicate that residues 315 and 317 are mutation hotspots in dasatinib-resistant pts, according to the experimental observation that they are both involved in inhibitor binding. They also provide a proof of principle that novel, inhibitor-specific mutant variants (i.e., T315A, F317I, K356R) may be selected, and raise some concerns about the limitations of single-agent treatment in the long term disease control of advanced phase-CML or Ph+ ALL pts. Supported by European LeukemiaNet, AIL, AIRC, FIRB and PRIN projects. Table 1 Pt Disease Mutation(s) before dasatinib start Best HR Best CgR Months on dasatinib Mutation(s) at relapse NE, not evaluated Primary resistance 1 CML/AP WT NR none 4 T315I 2 CML/AP T315I NR NE 1 T315I 3 CML/myBC T315I NR NE 1 T315I 4 CML/myBC F317L NR none 3 F317L 5 CML/lyBC T315I NR NE 1 T315I 6 Ph+ ALL T315I, M351T, L387M NR NE 2 T315I, M351T, L387M 7 Ph+ ALL T315I NR NE 1 T315I 8 Ph+ ALL F359V NR NE 2 T315I Acquired resistance 9 CP WT CHR minor 15 T315I 10 CML/myBC G250E NEL none 8 F317L 11 CML/lyBC Y253H CHR complete 9 CHR T315I 12 CML/lyBC WT CHR complete 4 T315I 13 CML/lyBC E255K CHR none 3 E255K, T315I 14 CML/lyBC D276G CHR complete 7 T315I 15 CML/lyBC WT CHR partial 9 F317L 16 Ph+ ALL E255K CHR NE 4 T315I 17 Ph+ ALL Y253H CHR complete 13 T315A, F317L, D276G 18 Ph+ ALL M351T CHR complete 13 M351T, F317L 19 Ph+ ALL WT CHR complete 6 F317I 20 Ph+ ALL WT CHR complete 4 K356R

We planned a prospective analysis involving 3 multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche Adulto) CML Working Party (WP) to evaluate the correlation between conventional cytogenetics (CC) and FISH response in CML pts in chronic phase (CP) treated with Imatinib. Karyotype and FISH analyses were performed on bone marrow (BM) cells in 26 local laboratories and in 12 WP reference labs. Peripheral blood samples for quantitative RT-PCR were centralized in Bologna. The data are reported in Figure 1 and in Table 1. Fig 1 shows the relationship between CC and FISH (r=0.91;p=0.008). Tab 1 shows the demonstration of FISH data according to CC data and the number of metaphases available for CC. Of 217 pts in CCgR by CC and > 20 metaphases studied, 81.6% were FISH negative, 15.2% showed a low rate of FISH positive cells (1–5%) and 3.2% an higher rate. Of 94 pts in CCgR by CC but with < 20 metaphases studied, 71.3% were FISH negative, 21.3% showed a low rate of FISH positive cells (1–5%) and 7.4% an higher rate. Of 43 pts in PCgR by CC and FISH positive, 48.8% showed 1– 5% positive cells. Moreover, 358 samples were performed simultaneously by CC, FISH and quantitative RT-PCR: 179 (50%) samples in CCgR showed major molecular response (MMolR, defined as a BCR-ABL × 100 ratio < 0.1%): 164 (91.6%) were FISH negative and 15 (8.4%) were FISH positive (1.3–10% positive cells). We conclude that interphase FISH is a very releable method of monitoring the CCgR once it has been achieved. The relationship of FISH on BM cells with molecular response is at least as good as the relationship of CC with molecular response. It remains to be shown if the same results can be obtained on peripheral blood cells, that are already widely used for molecular monitoring. | | No | FISH negative | FISH 1–5% | FISH > 5% | |:-------------- | --- | ------------- | ---------- | ---------- | | CCgR ≥ 20 met | 217 | 177 (81.6%) | 33 (15.2%) | 7 (3.2%) | | CCgR < 20 met | 94 | 67 (71.3%) | 20 (21.3%) | 7 (7.4%) | | PCgR ≥ 20 met | 43 | | 21 (48.8%) | 22 (51.2%) | | CCgR and MMolR | 179 | 164 (91.6%) | 12 (6.7%) | 3 (1.7%) | ![Figure][1] Figure [1]: pending:yes

Imatinib 400 mg (SD) is the established first line treatment of chronic myeloid leukemia (CML) in chronic phase. The efficacy of imatinib in early chronic phase has been demonstrated by phase 2 and 3 controlled trials like the IRIS study (O’ Brien et al NEJM 348:11, 2004). Large multicentric studies aimed to evaluate the impact of imatinib 400 mg outside strictly monitored frameworks are not yet available. The GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party opened in January, 2004, an observational study (serial n. CML/023) to investigate the efficacy of imatinib SD in newly diagnosed CML pts. Clinical and anagraphical data were collected through a web-based system. Peripheral blood samples for quantitative molecular analysis (RT-Q-PCR, Bcr-Abl/Abl × 100 - Taqman) were centralized in Bologna. Overall, 55 italian centers enrolled 367 (359 evaluable) newly diagnosed CML pts in chronic phase between Jan 2004 and Jan 2006. Median age was 50 yrs (range 18–84), 220 male and 139 females. Sokal risk at diagnosis was low, intermediate and high in 221 (62%), 123 (34%), 15 (4%) pts, respectively. 359 pts are evaluable for response at 3 months, 310 at 6 months and 187 at 12 months. The median observation time is 12 months. At 3 months, 94% of the pts reached a stable CHR. At 6 months, 80% of evaluable cases obtained a complete cytogenetic response (100% Ph-neg, CCgR). A major molecular response (MMolR) defined as a Bcr-Abl/Abl × 100 ratio < 0.1%, was shown in 52% of CCgR pts. At 12 months, the CCgR rate was 87% and the MMolR rate in CCgR pts was 63%. At 12 months, 3% of CCgR cases showed an undetectable level of transcript (ratio Bcr-Abl/Abl × 100 < 0,0001). With this short observation period, only 4 pts (1,1%) progressed to accelerated/blastic phase. Limiting the observation to low Sokal risk, at 12 months 221 such pts got a CCgR and MMol rates of 88% and 62%, respectively; 201 low Sokal risk pts were enrolled in the IRIS trial: at 12 months CCgR and MMolR (reduction of Bcr-Abl/Bcr ratio level > 3 logs) rates were 76% and 66%, respectively ( (T Hughes et al, NEJM 349:15, 2003). This study confirns that imatinib is efficacious and manageable, confirming and improving the results of the IRIS trial.

Extensive submicroscopic deletions adjacent to the breakpoint on derivative chromosome 9 [der(9)] have been reported in a subset of Chronic Myeloyd Leukemia (CML) patients and have been associated with an adverse outcome with conventional drugs and α-interferon (α-IFN). Huntly et al (Blood.2003; 102.2205–12) reported 275 CML pts who were treated with imatinib in CP, suggesting that der(9) deletions were associated with lower response rates and a shorter time to progression. Different data were reported by Quintas-Cardama et al (Blood.2005; 105:2281–6), who did not find any difference related with der(9) deletions in other 320 patients treated with imatinib. In these 2 studies, some patients began imatinib in early CP (51 and 152, respectively) while many patients (224 and 168, respectively) were treated in late CP. To establish the relationship of der(9) deletions with the response to imatinib in early CP patients, we planned a prospective study involving 3 consecutive multicentric national studies of the GIMEMA (Gruppo Italiano Malattie Ematologiche dell’Adulto) CML Working Party. 421 CML patients in early chronic phase were enrolled between January, 2004 and January, 2006; Fluorescence in situ hybridization (FISH) analysis of bone marrow cells was performed using BCR/ABL extra-signal, D-FISH or dual-color dual-fusion probes. At diagnosis, 52 (12%) of them had der(9) deletion and 369 (88%) had not. The 2 groups, with/without deletions, were comparable (no difference in age, Sokal risk, imatinib dose). Median observation time is 12 months. At 3 months, the CHR rates in with/without deletions patients were 87%/92%. At 6 months, the complete cytogenetic response (0% Ph-pos; CCgR) rates were 80%/80%, with major molecular response (MMolR, defined as a Bcr-Abl/Abl x 100 ratio < 0.1%) rates of 52%/51%, respectively. At 12 months, CCgR rates were 89%/86% and MMolR 52%/61%. No difference is significant. We conclude that the presence of der(9) deletions at diagnosis do not constitute a negative factor for response to imatinib: the haematological, cytogenetic and molecular response rates resulted equal between the 2 groups of patients with and without der(9) deletions. This finding is relevant to the long term effect of imatinib treatment, since both the CCgR and the MMolR are important and established indicator of long term survival.

We sought to determine the differences in molecular response between early and late CP pts with CML who achieved a CCR after treatment with IM at the standard dose of 400mg/d. We studied 2 different cohorts of patients in CCR: 67/191 (35%) pts after α-Interferon (α-IFN) failure enrolled on the CML/002/STI571 protocol 53/76 (70%) pts treated front line with a combination of IM and pegilated IFN-α (PEG-IFN) enrolled on the CML/011/STI571 protocol Cytogenetic response was monitored on bone marrow (BM) metaphases and molecular response was assessed by real time RT-PCR (TaqMan) BM and peripheral blood (PB) samples, collected at baseline, 3, 6, 9 and 12 months during the first year, and every 6 months thereafter. Molecular response was expressed as the ratio between BCR/ABL and β2-microglobulin (β2-M) x100. The lowest level of detectability of the method was 10−5. Negative results (i.e. undetectable transcript) were confirmed by nested PCR performed 4 times (sensitivity 10−6). For the purpose of this analysis, a major molecular response (MMR) was defined as a BCR-ABL/β2M value We observed a progressive decrease of the amount of BCR/ABL transcript in pts who achieved a CCR. At 24 months the median reduction in BCR/ABL transcript level was: a 3-log reduction in late CP pts a 4-log reduction in early CP pts In the latter group of pts MR was assessed also at 36 months. So we observed that 36 months after the first dose of IM and PEG-IFN pts who were still in CCR had the median value of BCR/ABL transcript of 0.00001% both in BM and PB. Therefore all these pts achieved a MMR. However only 8/53 (4%) pts were in CMR (undetectable BCR/ABL at least once as assessed by nested PCR). We conclude that front-line treatment with IM results in a better quality MR (4-log reduction in BCR/ABL transcript levels in early CP pts, as against a 3-log reduction in late CP pts). Figure Figure

To asses cytogenetic pattern of early diagnosed chronic phase chronic myeloid leukemia (CML) patients and to evaluate the role of either conventional (CC) and molecular cytogenetics in three multicentric studies, karyotype and interphase fluorescence in situ hybridization (FISH) analyses were performed in 372 enrolled patients between April 2004 and July 2005 by the GIMEMA CML Working Party (WP). Local investigator laboratories (25 labs) or WP reference labs (12 labs) performed both analyses. Cytogenetic examinations was performed at baseline; after 6 and 12 months of imatinib treatment; thereafter every 6 months and in case of failure or disease progression. At the baseline, 257 patients have been studied and 237 (92%) are valuable for both analyses (CC and FISH). Additional abnormalities in Ph+ clone have been observed in 12 patients (5%). Moreover, 18 (8%) cases showed variant Ph translocations and in 23 (10%) patients the derivative of chromosome 9 was deleted. As yet, cytogenetic response (CR) was evaluated in 188 samples and 156 cases were valuable (83%): 20 at 3 months, 101 at 6 months and 35 at 1 year of treatment. One hundred and eighteen (76%) patients achieved complete CR (CCR) established with more than 20 metaphases in 84 cases, meanwhile in 34 CCR cases the number of examined metaphases was lower. In the first group, 70/84 (83%) samples showed absence of bcr/abl rearrangement in FISH, meanwhile 13/84 (16%) carried a low rate of positive cells (1–5%) and the last one showed the rearrangement in 12% of cells. In the latter group, 23/34 (68%) didn’t show any rearrangement in FISH, in 8/34 (24%) the amount of Ph+ cells was low (1–5%), in 2 was higher (7% and 10%) and the last one carried an high rate (72%) of rearranged cells. In this latter case the RCC was evaluated on 10 metaphases. Twenty-three patients in major CR (MCR), but not in CCR, showed retention of persisting Ph+ cells ranging from 2 to 21% in CC study and from 2 to 16% in FISH analysis. Moreover we found a patient with 2% of Ph+ metaphases and 53% of Ph+ cells in FISH: in this case the CC evaluation was established with 10 metaphases. We can suggest there was a good correlation between cytogenetic and FISH tests in terms of the kinetics of disappearance of the bcr/abl rearrangement. FISH is a reliable method to reveal submicroscopic deletions and to monitor the size of the Ph + clone in treated CML patients. However, a good CC analysis remains an excellent approach to the evaluation of response to Imatinib. Moreover it can detect the emergence of other abnormalities in Ph positive or negative clone.

Using a novel denaturing-high performance liquid chromatography (D-HPLC)-based screening method, we investigated the frequency and prognostic relevance of ABL mutations in 47 CML patients who never achieved a major cytogenetic response in 12 months of Imatinib therapy. Seven patients were enrolled in the CML/011/STI571 trial (early-CP patients treated with Imatinib 400 mg/d and peghilated-interferon) while the remaining 40 were enrolled in the CML/002/STI571 trial (late-CP patients resistant/refractory to α-interferon, treated with Imatinib 400 mg/d). For each patient, a longitudinal analysis was done on all the available samples collected from Imatinib start up to the twelfth month of therapy. A nested RT-PCR was set up and, for each sample, two fragments of 393 and 482 bp spanning the ABL kinase domain were analysed. Two out of 7 (29%) early-CP patients had mutations, resulting in both cases in novel aminoacid substitutions (F311I, E355D). Mutations were already detectable at 6 and 9 months, respectively, from Imatinib start. Nineteen out of 40 (48%) late-CP patients had mutations. Eleven patients showed mutations falling in close proximity (M244V) or within (G250E, Y253F, Y253H, E255K, E255V) the P-loop. Eight patients showed mutations outside the P-loop (F311L, F317L, M351, E355G, F359V, H396R and a silent mutation at codon 298). Mutations were already detectable after a median of 3 months (range, 1–6) from onset therapy. At the time of mutation detection, all patients but four had sustained hematologic response. Presence of a missense mutation was significantly associated with a greater likelihood of subsequent progression to accelerated phase/blast crisis (AP/BC)(P=0.0002) and shorter survival (P=0.001). Patients with mutations falling in close proximity or within the P-loop had a particularly poor outcome in terms of time to progression to AP/BC with respect to the remaining mutated patients (P=0.03). Our results indicate that: i) ABL kinase domain mutations may be found also in the setting of early-CP CML patients, even though they are not the predominant mechanisms of resistance; ii) as many as 50% of late-CP patients who fail to achieve cytogenetic control of the disease in the first 6 months of therapy already have evidence of a mutation by D-HPLC analysis; iii) within cytogenetic nonresponders, presence of ABL mutations may identify a subset of patients with particularly poor prognosis. Thus, irrespective of the hematologic response, regular monitoring for emerging mutations in the first months of Imatinib administration may play a crucial role in detecting patients for whom a revision of the therapeutic strategy should be considered.

Older age constitutes a poor prognostic variable in CML patients: the negative effect of age on long-term survival has been consistently observed with most effective therapeutic modalities, both drug therapies (busulfan, hydroxyurea and interferon) and allogeneic transplant. In particular, older patients treated with interferon experienced much more adverse events than younger ones. In part their poorer prognosis was probably due to poor treatment compliance and few older patients have been included in prospective studies of interferon. Actually, imatinib is the first-line treatment for CML: its efficacy is very high and it seems to be well tolerated across age groups. We performed a sub-analysis of the effects of age on response and tolerance within the phase II trial of the italian GIMEMA CML Working Party (serial n.CML/002/STI571), which included 284 late chronic phase patients, treated with imatinib (400 mg daily) after interferon failure. Following the WHO, who defines “old” a patient ≥ 65 years, we analyzed the safety and efficacy of imatinib by age group: 226/284 patients (80%) were < 65 years (extr. 17–64) and 58/284 (20%) were ≥ 65 years (extr. 65–85 yrs) old. No significant differences between the two age groups were present at enrollment. Table 1 shows the responses and incidence of hematologic and non-hematologic adverse events (AEs). As expected, older patients experienced more AEs, both hematologic and non-hematologic. In part this significantly poorer tolerance probably justifies the lower response rates, both hematologic and cytogenetic. However, the overall survival was not different between the two age groups: with a median observation time of 33 months, the overall survival curves were superimposed (91%). Moreover, the rate of progression to accelerated/blastic phase was the same (10%). This study demonstrates that imatinib is greatly effective in older CML patients in late chronic phase (53% of MCR and 36% of CCR) and allows a good long-term survival. It is conceivable that treating old CML patients at the onset of the disease, as already shown by Jorge Cortes et al (Cancer2003;98:1105), could translate into response rates as the ones of younger patients. Responses and adverse events ≥ 65 (n. 58) ≤ 65 (n226) p value Complete Hematologic response 91% 99% 0,001 Major Cytogenetic Response 53% 74% 0,003 Complete Cytogenetic Response 36% 57% 0,001 Grade III Hematologic AEs 72% 50% 0,002 Grade III+IV Hematologic AEs 86% 60% 0,0001 Grade II non Hematologic AEs 83% 68% 0,0001 Grade III + IV non Hematologic AEs 29% 10% 0,0001

Background: Due to the BCR/ABL chimeric protein expression in chronic myeloid leukemia (CML), several genes and signaling pathways have been reported to be activated. These alterations in gene expression contribute to the pathophysiology of p210BCR/ABL-mediated transformation. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the mitogen-activated protein kinase or the ubiquitin phatway, whereas overexpression of other genes may implicate new cellular pathways involved in CML. The characterization of new genes which could be implicated in the pathophysiology of CML, may lead to the identification of potentially novel therapeutic targets for CML. In a previous study we adopted a microarray analysis to study the gene expression profiling of CD34+ cells taken from 5 de novo CML patients and treated in vitro with Imatinib (data not shown). Five genes had shown a greater down-regulation and a possible involvement in the in vitro effects of Imatinib. Aims: Aim of the present study was to confirm these data in vivo through a quantitative real time PCR analysis in 10 CML patients enrolled in a multicenter clinical trial of the GIMEMA CML working party (newly diagnosed, early-CP CML patients treated with Imatinib 400mg/die and peghilated interferon). Five patients were known to have chromosome 9 deletion. Methods: this study was performed on 10 bone marrow samples from 10 consecutive patients enrolled in the protocol. Bone marrow bone was collected prior to treatment (baseline) and after 3 and 6 months of Imatinib therapy. Five genes (TOPK, PBX3, SRPK, DDX21 and CLC) were analyzed at the RNA levels by means of a quantitative RT-PCR analysis (TaqMan). β2 microglobuline was used as control gene. Results were expressed as a median ΔCt value. Expression of these 5 genes before treatment with Imatinib and after 6 months of therapy was compared. Statistical significance was tested by the student’s T-Test. Results: Quantitative RT-PCR analysis confirmed that the 5 tested genes were downregulated after 6 months of treatment with Imatinib. About 1 log reduction was observed. The differences between the baseline median values and median values after 6 months of treatment were statistically significant (p>0.005). We were not able identify any differences between patients who showed chromosome 9 deletion and patients who did not. Conclusions: treatment of CML patients with the ABL-specific tyrosine kinase inhibitor Imatinib decreased expression at the mRNA level of all the genes tested. This suggests that increased gene expression could be in some case tyrosine-kinase dependent and it could be implicated in the pathophisiology of CML. Study supported by: COFIN 2003 (M. Baccarani), AIRC, AIL, Fondazione del Monte di Bologna e Ravenna, FIRB 2001.

CML is a clonal myeloproliferative disease which typically presents in chronic phase (CP), in which malignant progenitor cells proliferate rapidly but retain much of their ability to differentiate, with the disease later evolving to accelerated phase/blast crisis. Even after the introduction of imatinib, the calculation of the Sokal and the Euro prognostic scores has remained essential in clinical practice, since allow to stratify CML patients at different evolutive risk at diagnosis, guiding therapeutic decisions. More recently, numerous research efforts are ongoing to gain a better understanding about the intrinsic heterogeneity of CML, in order to identify a novel molecular signature which might characterize patients (pts) with different prognosis, and propensity to respond to treatment In the present study we adopted a GEP strategy in an attempt to identify genes and pathways, able to predict and/or elucidate the disease course of CP-CML pts at the time of diagnosis. To this aim, highly enriched CD34+ cells from peripheral blood obtained at diagnosis from pts with untreated Ph+ CML in CP were used throughout the study. Overall, 28 pts were included in the present analysis. They were diagnosed from August 2006 to June 2008; front-line treatment was either imatinib (12 pts) or nilotinib (16 pts). GEP was performed using the Affymetrix HG133 Plus microarray platform as per manufacturer’s recommendation. Raw data was normalized using the RMA algorithm and filtered. Unsupervised analysis was performed by Multidimensional Scaling and hierarchical clustering. Top differential genes were selected by significance analysis of microarrays method (SAM), setting the FDR threshold 0.01. Genes associated with Sokal risk score were searched by various methods (Limma, ANOVA, SAM, ROC analysis, EB-arrays). Analyses were performed using R and Bioconductor, BRB array tools and MeV. To analyse the data, we have first stratified our cohort of pts according to their Sokal risk, into 3 groups (high, intermediate and low risk) of 10, 13 and 5 pts, respectively. We did not identify any significant gene signature uniquely associated with Sokal risk score, even by various supervised techniques. Neither was it possible to obtain a signature from the most extreme phenotypes, i.e. by excluding intermediate-risk pts. Interestingly, however, unsupervised analysis of the whole gene expression data set clearly identified two subgroups of pts (we have named them A and B), which included 15 and 13 pts, respectively. Those two subgroups of pts show a differential expression of a list of at least 461 probe sets, which represents the most significantly up and down regulated probes, without any false positive. Both a hierarchical clustering of these probe sets, as well as a multidimensional scaling plot showed a clear demarcation between the two subgroups of pts, and the Sokal risk score did not co-vary along with their gene expression profile. In the group A of pts, of the 461 probe sets, 317 resulted up-regulated and 144 down-regulated. Genes up-regulated are mainly involved in regulation of transcription and/or gene expression (40/317 probe sets code for zinc finger protein), whereas down-modulated genes are involved in cell differentiation, cell death and cell cycle regulation. Of interest, several genes and functional classes showed the same deregulated expression as previously described during progression to blast crisis of CML (Radich et al. PNAS 2006). Moreover, 6 probe sets (EREG, FOS, IL8, WT1, HSPA1A and DKFZp761P0423) that are part of the Radich “top ten” list of genes most significantly associated with progression in CML, are differentially expressed in our two subgroups. Overall, our data suggests the existence of two biologically distinct subgroups of CML, irrespective of Sokal score, which could be identifiable at diagnosis. Those findings further support a hypothesis that different clinical behaviours of the disease and response to treatment could be associated with different gene expression profiles of individual pts. Our plan is to follow up the pts which we have analyzed in this study and to investigate if those molecular signatures, identified through GEP could be correlated with treatment responses.

Abstract 3262 Poster Board III-1 CML pts display a certain degree of clinical heterogeneity that is documented by the varying levels of response to tyrosine kinase inhibitor therapy and is best reflected by the Sokal risk score. Clinical differences must be a sign of some biological heterogeneity the basis of which, however, are still poorly understood. Today many high-throughput assays are available that allow to unravel the complexity of cancer cells in a genome-wide fashion. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution ( The pathways and genes most frequently affected by CNAs or UPD are listed in the Table below: Base Excision Repair (BER) MUTYH DNA glycosilase 1p34.1 loss/upd 10 pts PNKP Polynucleotide kinase 19q13.33 loss 10 pts NEIL1 DNA glycosilase 15q24.2 loss 7 pts POLB DNA polymerase beta 8p11.21 loss/upd 6 pts PCNA Sliding clamp for DNA polymerases 20p12.3 loss 6 pts Mismatch Repair (MMR) PMS2L5 Mut L homolog - mismatch and loop recognition 7q11.23 loss/upd 11 pts MSH2 Mut S homolog - mismatch and loop recognition 2p21 loss 8 pts POLD1 DNA polymerase delta 19q13.33 loss 8 pts POLA2 DNA polymerase alpha, subunit 2 2p16.3 loss/upd 6 pts POLE2 DNA polymerase epsilon, subunit 2 14q21–22 loss 5 pts Nucleotide Excision Repair (NER) ERCC1 5’ incision subunit of TFIIH complex 19q13.32 loss 11 pts ERCC2 5’ to 3’DNA helicase of TFIIH complex 19q13.32 loss 10 pts XAB2 Transcription-coupled NER factor 19p13.2 loss/upd 9 pts CDK7 Kinase subunit of TFIIH complex 5q13.2 loss 7 pts RPA4 Binds damaged DNA in preincision complexes Xp21.33 loss/upd 7 pts RPA2 Binds damaged DNA in preincision complexes 12q24.31 loss/upd 6 pts Homologous Recombination (HR) RAD51C Homologous pairing 17q23.2 loss/upd 7 pts RAD52 Accessory factor for recombination 12p13.33 loss 7 pts XRCC2 DNA break and crosslink repair 7q36.1 loss 5 pts Non-Homologous End Joining (NHEJ) PRKDC DNA-dependent protein kinase, catalytic subunit 8q11.21 loss/upd 5 pts DCLRE1C Artemis nuclease 10p13 loss 5 pts REV7 DNA polymerase zeta, subunit 1p36.22 gain 5 pts Other genes involved in DNA replication/repair/modification or chromatin remodeling CHAF1A Chromatin assembly factor 19p13.3 loss 15 pts RECQL5 DNA helicase 17q25.1 loss/upd 11 pts RAD9B PCNA-like DNA damage sensor 12q24.11 loss/upd 10 pts CHEK2 DNA-damage checkpoint 22q12.1 loss/upd 8 pts RAD17 RFC-like DNA damage sensor 5q13.2 loss 6 pts TREX1 DNAase III exonuclease 3p12.31 loss/upd 6 pts For some genes (e.g., RAD52), the monoallelic deletion we detected was found to translate into reduced mRNA expression, observation that was also independently confirmed in an additional group of high/intermediate versus low Sokal risk pts. In all the 44 pts, multiple pathways and multiple genes within the same pathway were affected, supporting the hypothesis that the lesions we detected might actually have consequences on DNA integrity despite the known partial functional redundancy of pathways and effectors. For many of the genes identified in this screen, activating or inactivating mutations are known to occur, and together with overexpression or haploinsufficiency, have been linked to a mutator phenotype in several malignant conditions. We are currently investigating whether this may be the case also in CML. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: Baccarani: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.