ALBERT

Description: Background and rationale In chronic myeloid leukemia (CML) about half of patients (pts) achieving a deep and stable molecular response (MR) with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria to possibly identify more and better CML patients suitable for an efficacious discontinuation are today focus of research in CML. Leukemia stem cells (LSCs) are supposed to be the reservoir of disease. We first showed in a cross-sectional study including 112 pts in TFR for a median of 31 months (mos) that residual circulating CD34+/CD38-/CD26+ CML-specific LSCs were still detectable in the majority of CML pts despite stable and deep molecular response. This evidence suggested that the level of BCR-ABL transcript only may not reflect the actual residual CML LSCs burden and that there could be a "threshold" of LSCs predicting a successful TFR. Aims To further study the behavior of residual LSCs during TKI discontinuation we designed a prospective multicentered study (AIRC IG 20133 study) in which we monitored circulating CD26+ LSCs in CML pts from the time of TKI discontinuation until molecular relapse. Methods CML pts meeting the current molecular criteria for TKI withdrawal entered this multicenter study. At TKI stop (baseline) and at +1, +2, +3, +6, + 12 mos after discontinuation and at any time if molecular relapse, CML pts were evaluated for peripheral blood number of CD34+/CD38-/CD26+ LSCs by centralized flow-cytometry analysis and for BCR-ABL transcript level by standard (IS) quantitative RT-PCR assay. Results 49 consecutive CML pts were enrolled in the study so far. Pts characteristics at diagnosis, type of TKI, disease response and treatment duration before discontinuation are shown in Tab. 1. After a median time of 7 mos since TKI stop (range 1-24), 13/49 (26.5%) pts lost their molecular response and restarted TKI treatment. Median time to relapse after discontinuation was 4 mos (range 2-7). 36/49 (73.4%) pts are still in TFR after a median time of 7.5 mos (range 1-24). If considering a cut-off of 6 mos from discontinuation as the period with higher risk of relapse, 14/36 pts actually in TFR have discontinued treatment for ≤ 6 mos (range 1-6) while 22/36 pts are in TFR for a median of 10 mos (range 7-24). Regarding residual CML LSCs evaluation, at baseline 23/49 (46%) pts had still measurable circulating CD26+LSCs with a median number of 0.0204µ/L (range 0.0077-0.1197), while 26/49 (54%) had no detectable CD26+ LCSs. Considering the small number of molecular relapses no statistical difference in number of residual CD26+ LSCs at time of discontinuation was shown between pts losing vs maintaining TFR (13 pts median CD26+ LSC 0.0237/µ/L, range 0-0.1197 and 36 pts median CD26+ LSCs 0.0204/µ/L, range 0-0.1039, respectively). However, the number of pts with undetectable CD26+ LSCs at baseline was 6/13 (45%) and 20/36 (55%) in the two subgroups, respectively. Considering subsequent time points, the 13 relapsed pts showed a small yet progressive increase of residual CD26+ LSCs number until molecular relapse, while the 36 pts in TFR showed a fluctuation of CD26+ cells number. However, Kendall rank correlation coefficient, Mood test and bi-linear relation model of the whole cohort showed no correlation between BCR-ABL/ABLIS ratio and number of residual CD26+ LSCs either at baseline or at each time points after discontinuation, thus confirming our previous observations. Conclusions Yet very preliminary our results showed that CD26+ LSCs are detectable at time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study (7.5 mos) the persistence of "fluctuating" values of residual CD26+ LSCs do not hamper the possibility to maintain a stable TFR. Due to the short follow up and the small number of molecular relapsed pts we could not find a threshold of CD26+ LSCs predictive of TFR loss. Our data may suggest other factors then LSCs "burden" to play an active role in controlling disease recurrence. Additional studies evaluating CD26+ LSCs ability to modulate the immune system through a variable expression of immune response inhibitory molecules and through their interactions with effectors cells are ongoing. Table Disclosures Bocchia: Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Abruzzese:Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Iurlo:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Galimberti:Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Liberati:Bristol & Mayer: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria.

Description: The prognostic significance of the response to initial prednisone treatment in adult ALL has been recently emphasized. Prednisone response is usually defined on the basis of the peripheral leukemic blast count. The threshold value for the defintion as good or poor prednisone response is 1000 blasts/mmc on day 8 of prednisone pre-treatment. The drawback of this definition is the difficulty of classifying patients with less than 1000 blasts at diagnosis. In the LAL2000 GIMEMA study we therefore evaluated whether the blast reduction rate, which is not affected by the initial blast level, could be a factor with comparable prognostic value. The protocol design provided a 7-day (−6 to 0) pre-treatment phase with an escalating dose of prednisone up to 60 mg/sqm. On day 1 before starting the induction the response was assessed both according to the absolute blast count (〈 versus ≥ 1000/mmc) (criterion 1) and according to the blast reduction rate ≥ 75% (criterion 2) in the peripheral blood. The induction included high dose Daunorubicin; for patients in complete remission (CR) this was followed by consolidation with high dose ARA-C, chemo and radio prophylaxis of the central nervous system, and periodical reinduction over a three years maintenance period. Patients with adverse cytogenetic features [i.e. t(9;22), t(4;11), t(1;19)] who achieved a CR were treated according to the HAM protocol that included high dose ARA-C and Mitoxantrone followed by Imatinib for Ph+ ALL and by allogeneic or autologous hemopoietic stem cells transplantation for the others. Between September 2000 and December 2003 a total of 368 patients were evaluable for response to induction. The median age was 35 years (15–60) and median WBC count 15′109/L (0.3–872); 72 (20%) were T ALL and 121 (33%) had cytogenetic high risk features (104 (86%) Ph+, 4 (3%) t(4;11) and 13 (11%) t(1;19)). Eighty-seven percent of the patients were evaluable for response to steroid pre-treatment: ’responders’ were 75% according to criterion 1 (blast

Description: G-CSF is used to enhance neutrophil recovery after autologous peripheral blood stem cell transplantation (aPBSCT), even if the optimal timing and dose of G-CSF has not been established. Recently Peg-Filgrastim has been approved for clinical use. The clearance of Peg-Filgrastim is neutrophil-mediated with a sustained duration of action with prolonged serum concentration during neutropenia. In order to verify the efficacy and feasibility of use of Peg-Filgrastim, we administered to 10 patients (A Group, 3F, 7M, median age 46y) submitted to aPBSCT for haematological malignancies (6 NHL, 3 MM, 1 HD) a single 6 mg dose of Peg-Filgrastim subcutaneously on day +1 and we compared the engraftment of these patients to data of a group of 30 patients (B group 9F, 21M, median age 46y, 18 NHL, 6 MM, 2 POEMS syndrome, 1 Plasmacell leukemia, 3 HD) submitted to aPBSCT not receiving G-CSF. Patients were matched for sex, age, disease, disease status at transplant, conditioning regimen, transplant procedure and they received comparable CD34+ cell dose. We evaluated haematological engraftment, and other clinical outcomes, all results are expressed as a median value in the table. Peg-Filgrastim was well tolerated in all but 1 patient reporting bone pain. Neutrophil engrafment was significantly faster (72 hours) in the Peg-Filgrastim group: days +10 and +12 (500 and 1000 cells/μL) vs days +13 and +15.5 of control group; faster neutrophil engraftment was also clear analyzing number of days with absolute neutrophil count (ANC) 500/μL 10 (7–11) 13 (7–18) p 1000/μL 12 (7–15) 15.5 (10–38) p 20 x 10³/μL 11 (8–24) 12 (9–18) p=0.4 Days to Plts 〉 50 x 10³/μL 24 (12–39) 14 (10–60) p=0.39 Days to Plts 〉 100 x 10³/μL 40 (14–120) 18 (12–210) p=0.06 Days to Reticulocytes 〉1% 15 (11–16) 13.5 (10–30) p=0.59 Days to Hgb 〉10 g/dl 33 (10–84) 21 (10–210) p=0.48 Days of fever 5 (2–10) 5 (2–13) p=0.92 Days of antibiotic therapy 7 (5–20) 9.5 (5–18) p=0.72 Bloodstream infections 4/10 6/30 p=0.2 Number of pRBCu 1 (0–2) 0 (0–2) p=0.02 Number of SDu 1.5 (1–3) 1 (0–2) p=0.022 Days of hospitalization 25 (19–30) 23 (20–34) p=0.89

Description: 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

Description: Abstract 2279 Over the years, Bcr-Abl kinase domain (KD) mutation analysis has been more and more extensively applied in Philadelphia-positive (Ph+) patients (pts) resistant to tyrosine kinase inhibitors (TKIs) to assist clinicians in therapeutic decisions. We reviewed the database recording the results of mutation analyses performed in our laboratory from January 2004 to June 2010. Overall, 2996 Bcr-Abl KD mutation screening tests were successfully performed by denaturing-high performance liquid chromatography (D-HPLC) and/or direct sequencing of the Bcr-Abl KD (residues 206–524). The total pts analyzed were 1139 (CML, n=1005; Ph+ ALL, n=134); the number of tests per patient ranged from 1 to 14. One hundred and ninety-one tests on 148 pts were performed in 2004, 391 tests on 214 pts in 2005, 469 tests on 217 pts in 2006, 521 tests on 241 in 2007, 536 tests on 301 pts in 2008 and 576 tests on 311pts in 2009. Overall, 869/2996 tests (29%) yielded a positive result. In 91/869 (10.5%) cases, D-HPLC showed evidence of sequence variations below the lower detection limit of sequencing; in the remaining 778 cases mutations could be characterized – a single mutation was detected in 646 (83%) cases, 2 mutations in 95 (12%) cases, 3 mutations in 25 (3%) cases, 4 or more mutations in 12 (2%) cases. In those pts for whom a longitudinal analysis was performed, 2 or more mutations accumulated as a consequence of multiple lines of therapy in 69% of cases, while in the remaining cases they concomitantly emerged under the same TKI. In 8 (10%) cases, small insertions or deletions were detected (Δ248-274 in 4 cases). Silent mutations were detected in 32 cases, either alone (11 cases) or in association with missense mutations. The K247R polymorphism was detected in 3 pts only. Five hundred and seventy-seven pts were analyzed at the time of resistance to imatinib (IM), 94 at the time of resistance to a 2nd TKI, 34 at the time of resistance to a 3rd TKI. In the IM-resistant setting, the ten most frequently detected mutations were F359V (13.4% of pts), M351T (12.3%), M244V (12.3%), H396R (10.3%), G250E (8.2%), E355G/D (8.2%), E255K/V (6.1%), Y253F/H (6.1%), T315I (4.1%), F317L (4.1%) in chronic phase (CP) CML pts; T315I (15.6%), G250E (13.7%), M351T (9.8%), E255K (7.8%), Y253F/H (7.8%), M244V (7.8%), Q252R/H (5.8%), H396R/P (5.8%), L384M (3.9%), F359V (3.9%) in myeloid blast crisis (BC) CML pts; and E255K/V (16.6%), T315I (16.6%), Y253F/H (15.2%), G250E (12.5%), M244V (8.3%), Q252R/H (5.5%), M351T (4.1%), L248V (2.7%), F359V (2.7%), D276G (2.7%) in lymphoid BC CML/Ph+ ALL pts. In the CP CML setting, 102 pts were analyzed because of strictly defined failure to 1st-line IM according to the 2006 ELN recommendations. Thirty-two (31%) were positive for mutations; only one had a T315I. Ninety-nine out of 128 (77%) CML and Ph+ ALL pts who were reported to be resistant to a 2nd or a 3rd TKI (either nilotinib or dasatinib) were positive for one or more mutations. The ten most frequent ones were T315I (30.3% of pts), F317L (16.2%), Y253H (16.2%), F359V (7.1%), V299L (7.1%), E255K (6.1%), E255V (5.1%), F359I (4%), T315A (3%), F359C (2%) – either alone (56% of pts), combined (29%), or together with other mutations (15%). Preferential associations between mutations were observed. Eighty-five CP CML pts on IM were analyzed because of increasing Bcr-Abl transcript levels, including 61 pts who experienced ≥1-log increase without loss of major molecular response (MMR) and 24 pts who experienced ≥1-log increase leading to loss of MMR (but not of complete cytogenetic response). Mutations were identified in 2/61 (3%) and 3/24 (12.5%) pts, respectively. Forty-four CP CML pts (Low Sokal, n=14; Intermediate Sokal, n=15; High Sokal, n=15) were screened for mutations at the time of diagnosis, including 21 pts who later relapsed with evidence of mutations. Only 1 High Sokal risk patient scored positive for a mutation at diagnosis (Y342C); at the time of relapse, however, the mutation had disappeared and an M244V was instead detectable. Fifty-five Ph+ ALL pts were analyzed at the time of diagnosis. D-HPLC showed evidence of mutations in 3 (5%) pts, but they were all below the lower detection limit of sequencing. All 3 pts later relapsed with KD mutations (T315I). Additional sub-analyses will be presented. Our seven-year experience in a large series of pts sheds further light on the frequency and clinical relevance of Bcr-Abl KD mutations in the IM and in the 2nd generation TKI era. Supported by ELN, AIL and PRIN. Disclosures: Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Description: Abstract 3460 Autoimmune diseases (AD) have been reported after haematopoetic stem cell transplantation (HSCT) and most commonly autoimmune cytopenias (AIC) are observed. Proposed risk factors are unrelated donor HSCT and presence of chronic graft-versus-host disease (GVHD). Use of CB cells is increasing as an alternative source of adults HSC for allotransplant however incidence and risk factors of AD after CBT have not been described. Since CB lymphocytes are immature and most of the CBT are HLA mismatched, we hypothesize a high incidence and diversity of secondary AD and. Therefore, we conducted a retrospective study in order to describe incidence, nature and risk factors for secondary AD after CBT. We include CBT recipients reported to EUROCORD up to December 2008. All centres were asked to participate to the survey even if they did not observe any case of AD after CBT. For those with AD, detailed information on diagnosis, treatment and outcome were asked. Median follow-up was 44 months (3.2-217). 42 out of 651 patients (pts) from 35 centres had developed at least one secondary AD at a median of 198 days (27-4267) after CBT. The remaining 609 pts without secondary AD served as control group (nonAD). Characteristics of both groups are given in table 1. Cumulative incidence of secondary AD after CBT was 5±1% after 3 years and 6.5±1% after 10 years. Diagnosis of secondary AD were autoimmune haemolytic anemia (AIHA) in 16, autoimmune thrombopenia (ITP) in 9, EVANS syndrome in 6, autoimmune thyroiditis in 3, glomerulonephritis in 2, Graves disease, psoriasis, psoriasis arthritis, immune neutropenia, ulcerative colitis and vasculitis in 1 patient each. Three pts developed ITP followed by AIHA. Median time to develop a AIC was 180 days (27-4267) and non-hematological AD was 322 days (70-1117), respectively (p=0.13). At onset of AD, 3 pts had acute GvHD, 6 chronic GvHD and in 10 pts an infection. In multivariate analysis adjusted for differences between the two groups, the following factors were associated with higher incidence of secondary AD: 1) age

Description: Abstract 3409 A score aiming at early identification of CML patients showing sensitivity to second generation TKIs was proposed by the Hammersmith group. The score was created by analizing 80 patients and was based on 3 prognostic factors: previous cytogenetic response to imatinib, Sokal risk and recurrent neutropenia during imatinib. Subsequently, the score was validated in a small series of 28 patients. Aim of our study was to confirm the validity of this score and to establish its strength on a large group of CML patients resistant to imatinib and treated with second generation TKIs. One hundred twenty-seven patients 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 received interferon before imatinib. Thirty patients had primary resistance, whereas 97 patients received second-generation TKI after acquired resistance to imatinib. The application of Hammersmith score was possible in 118 patients with available data: 52 patients were identified as good risk, 27 patients as intermediate risk and 38 patients as poor risk. The 1-year cumulative incidence of complete cytogenetic response (CCR) was 73% in good risk patients, 40% in intermediate risk patients and 23% in poor risk patients (p=0.0001). Similarly, the cumulative incidence of major molecular response (MMR) was 52% in good risk, 28% in intermediate risk and 13% in poor risk category (p=0.001). In the evaluation of event-free survival (EFS), events were considered loss of hematologic or cytogenetic response, disease progression, death for any cause, toxicity: the estimated 2-year event-free survival (EFS) was 89% in good risk, 70% in intermediate risk and 55% in poor risk group (p=0.0001). Progression-free survival (PFS) was defined as survival without evidence of accelerated or blastic phase: the estimated 2-year PFS was 97% in good risk, 93% in intermediate risk and 87% in poor risk category (p=0.05). Kaplan Meier estimated 2-year overall survival (OS) was 100% in the good risk, 93% in the intermediate risk and 82% in the poor risk category (p=0.001). In conclusion, as suggested by Milojkovic et al, some prognostic factors before starting second generation TKIs might predict cytogenetic response and outcome. As far as we known, the so-called Hammersmith score was not yet validated in large series of patients: we demonstrated that this score was able to discriminate patients at high risk of failure and consequent progression before treatment with second generation TKIs. Disclosures: No relevant conflicts of interest to declare.

Description: Tyrosine-kinase inhibitors (TKIs)have completely changed the expected survival of chronic myeloid leukemia (CML) patients which is now approaching that of the general population: a relevant proportion of CML patients are currently elderly or very elderly. Very elderly patients represent generally a small proportion in published experiences. Older CML patients imatinib treated, as it happens in the general population, receive other drug treatments for associated chronic illnesses. Our aim is to assess if and which classes of concomitant drugs have an impact on cytogenetic response in chronic phase (CP)-CML very elderly (age 〉75 years) patients. Two hundred and twelve very elderly CP-CML patients, imatinib treated at 33 italian hematological institutions have been retrospectively evaluated. Median age at diagnosis was 78.5 years (range 75.0-93.0); 111 (52.4%) were male. Sixty-two (29.2%) were Sokal high risk. Sixty-seven (31.8%) were treated with reduced dose imatinib (400 mg/day. Concomitant drugs were 1-2 in 73 (34.4%) patients, 3-4 in 59 (27.8%), and 〉5 in 64 (30.2%); 16 (7.6%) did not assume any concomitant drug. Drugs more frequently used were antiplatelets, assumed by 104 (49.1%) patients, followed by diuretics in 91 (42.9%) patients, proton pump inhibitors (PPIs) in 86 (40.6%), ACE inhibitors in 55 (25.9%), beta blockers in 44 (20.7%), angiotensin II receptors blockers (ARB) in 41 (19.3%), calcium channel blockers in 34 (16%), statins in 25 (11.8%), and alpha blockers in 11 (5.2%). Univariate logistic regression models were computed to assess the association between cytogenetic response after 6 or 12 months of imatinib treatment and number of concomitant drugs or selected drug classes. Statistical analyses were done using JMP 11.1 (SAS Institute Inc., Cary, NC, USA). Complete cytogenetic response (CCyR) was obtained in 124 (58.8%) patients, of whom 70 (33%) within 6 months. Consequently, we focused our study on the impact of number and types of drugs on CCyR rate, which represents the primary therapeutic endpoint in the elderly. Cytogenetic response distribution according to concomitant drugs is reported in table 1. We did not find any significant correlation between number of concomitant drugs, single classes of antihypertensive drugs, antiplatelets, PPIs or statins and CCyR rate at 6 or 12 months. Even though few pharmacokinetic interactions are reported between imatinib and some of medications we considered, this does not seem to have an impact on cytogenetic response rate in our cohort. Indeed, our results confirm the well-known safety and efficacy of imatinib also in very elderly CML patients. Table 1. Cytogenetic response according to concomitant drugs Drug classes Cytogenetic response CCyR 12 months No CCyR Antiplatelets (n=104) 38 (36.5%) 31 (29.8%) 11 (10.6%) 24 (23.1%) Diuretics (n=91) 32 (35.2%) 21 (23.1%) 13 (14.3%) 25 (27.4%) Proton pump inhibitors (n=86) 30 (34.9%) 22 (25.6%) 13 (15.1%) 21 (24.4%) ACE inhibitors (n=55) 19 (34.6%) 11 (20%) 12 (21.8%) 13 (23.6%) Beta blockers (n=44) 18 (40.9%) 11 (25%) 3 (6.8%) 12 (27.3%) Angiotensin II receptor blockers (n=41) 19 (46.3%) 11 (26.8%) 5 (12.3%) 6 (14.6%) Calcium channel blockers (n=34) 10 (29.4%) 7 (20.6%) 6 (17.7%) 11 (32.3%) Statins (n=25) 9 (36%) 7 (28%) 2 (8%) 7 (28%) Alpha blockers (n=11) 4 (36.4%) / 1 (9.1%) 6 (54.5%) Disclosures Castagnetti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Tiribelli:Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

Description: Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment. We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues: i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence; ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results; iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases. Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts: i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples. ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS. iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples. DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%. Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline. In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI. In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized. Conclusions MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs; polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing; the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse; MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.