Though the initial response to induction therapy, indicating rapid tumor clearance, is currently considered to be one of the most important prognostic factors, detection of submicroscopic levels of minimal residual disease (MRD), by means of PCR or immunophenotyping after induction and during different stages of treatment, has also become important in predicting outcome in ALL patients. We initiated a study using parallel PCR and flow cytometry to monitor MRD in adult acute lymphoblastic leukemia (ALL) patients treated by standard approach. Bone marrow samples were analyzed at diagnosis, after pre-phase (one week of steroid therapy to determine steroid responsiveness), at 8-weeks post induction, at consolidation, before maintenance (+7–8 mo), and during maintenance for the detection of clonal IgH, TCR-gamma rearrangements by PCR (The level of detection sensitivity is 10−3 to 10−4). Specific markers were identified for 88% of the patients (30/34); prolonged monitoring was carried out in 22 patients with follow up from 2 to 24 months. Fifteen of those patients were analyzed by three-color flow cytometry analysis with a panel of 20 monoclonal antibodies for patient-specific aberrant leukemia-associated phenotype (LAP) at diagnosis and before maintenance (10−4). Contrary to PCR analysis, LAPs were detected in all patients. The most common LAPs were CD19/34/10, CD 19/34/TdT, and CD19/34/IgM. Clinical CR was achieved in 85% of patients. Prednisone resistance was detected in 71% of patients. MRD was detected by PCR in all patients after pre-phase, in 21/22 (95%) after 1st induction phase, in 11/15 (73%) after 2nd induction phase, in 10/15 (67%) after consolidation, and in 8/14 (57%) before maintenance. Flow cytometry data coincides with molecular parameters: only 4 of 10 patients examined before maintenance (+7–8 mo) had no cells with LAP. Comparing our parallel measurements we have noted discrepancies in 3 of 10 cases. Two immunologically positive patients were PCR-negative, and one PCR-positive patient was immunologically negative. All patients (n=3) whose PCR probes were positive more than twice, relapsed at different time points; no relapses were noted in once positive patients (n=3), and 1of 8 PCR-negative patients relapsed. Our data indicate that both methods are effective and relevant in detection of MRD. They show very slow tumor clearance in ALL patients. MRD positivity reflects the higher relapse probability. More patients can be followed for MRD with flow-cytometry, due to higher detection rate of LAP.
2572 Adult ALL regardless recent advantages in adolescents and young adults is still considered to be a therapeutical problem. So called “a pediatric-like approach” applied in adult ALL is reported to be more reasonable even in the older adults (up to 55 y). RALL has initiated a prospective multicenter trial for adult Ph-neg ALL based on: 1) evaluation of b/m blast clearance after 7 days of Prednisolone (PRD) prephase and its substitution by Dexamethasone (DEXA) if b/m blast count was >25%; 2) continuous 2,5 years treatment schedule with prolonged L-asparaginase (Σ=590.000 IU); 3) evaluation of the impact of the late intensification (2 courses of HD of methotrexate and ARA-C) on MRD clearance. The study is registered on the ClinicalTrials.gov public site; NCT01193933. From Nov, 2008, till June, 2012, 24 centers enrolled 173 pts: median age 28 y (15–55), 71f/101m, 112 (64,7%) = B-lin, 54 (31,2%) = T-lin, 1 pt -undifferentiated AL (0,6%), 6 - uknown phenotype (3,5%), median LDH=848 ME (72–13061), median L=13,5 (0,6–556*109/l). Cytogenetics was evaluable in 58,3% of pts (n=101) and 46,5% of them (n=47) had normal karyotype (NK). Initial risk group was evaluated in 154 pts among whom 46 patients (29,9%) were in the standard risk (SR) group (WBC <30 for B-Lin, <100 for T-Lin, EGIL BII-III, T-III; LDH < 2N, No late CR, t(4;11)-negative), 109 (60,1%) - in the high risk (HR) group (WBC >30 for B-Lin, >100 for T-Lin, EGIL BI, T-I-II-IV; LDH > 2N, No late CR, t(4;11)-positive). 19/173 pts (11%) were not qualified. The analysis was performed in June, 2012. +8 day b/m blast count was reported in 149 pts and b/m blasts less than 25% were detected in 36,2% of pts. The portion of PRD non-responders was statistically different in SR and HR groups: 24/45 (53,3%) and 68/101 (67,3%) (p=0,01), confirming the initial risk groups identification. Induction results were obtained in 150 pts, and CR rate was identical in both risk groups (SR=86,9%; HR=84,1%) with total 12 induction deaths (8,0%) and 6 resistant leukemias (4,0%). With a median follow-up of 12 mo (1–36 mo) death in CR was reported in 9/132 (6,8%) pts. OS at 36 mo was 58,6%, DFS-68,3%. MRD analysis for clonal IgH and TCR rearrangements was carried out in 25 pts. And as in our previous studies (ASH 2006, abstr 2294) the clearance was slow with only 41,6% pts negative for MRD at day +133 (4mo) of the protocol. Two late intensification courses (day +157 = 5 mo) increased MRD negativity only up to 50%. Such slow MRD clearance did not correspond to higher relapse rate so far. Age, WBC, immunophenotype, LDH, risk group, +8 day b/m blast count, time to CR, time without treatment (<>8days), L-asparaginase cessation did not influence survival. OS and DFS differed in pts with NK vs all other abnormalities: 87,4% vs 57,9% (p=0,002) and 88,9% vs 66,5%, respectively (p=0,02). So, our data demonstrated that ALL-2009 protocol provided 58% 3-years overall survival and 68,3% DFS. In adult Ph-neg ALL normal karyotype predicts better survival. The MRD clearance is very slow while on this protocol. Disclosures: No relevant conflicts of interest to declare.
4895 Introduction: Minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) can be analyzed by flow cytometry, fluorescent in situ hybridization (FISH) if there are chromosomal abnormalities, polymerase chain reaction (PCR) for chimerical transcripts and immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRG) genes rearrangements. IgH and TCRG rearrangements are the for monitoring of MRD, because it is possible to find specific clonal tumor marker in 80–90% patients. According to the data of different research groups phenomenon of oligoclonality (more than one clonal rearrangement) is identified in 16–80% cases depending on how much molecular markers are used (Scrideli CA, 2001; Gameiro P, 2002; Thorsten Raff, 2006; Csinady E, 2009; Katsibardi K, 2011). Methods and materials: We studied bone marrow samples from 59 patients with new diagnosed B-lineage ALL (43 patients), T-lineage ALL (15 patients) and one patient with bilinear B-T-ALL. Ph-positive ALL was diagnosed in 6 patients, t(4;11) – in one patient. Patients were treated by standard ALL-protocols based on 8 weeks induction remission, 3–5 consolidation courses and maintenance treatment during 2 years. Patients with Ph-positive B-ALL were treated by the same chemotherapy with imanitinib and one patient with mature B-ALL was treated by NHL-BFM-90 chemotherapy. Detection of IgH and TCRG genes rearrangements were carried out by polymerase chain reaction using standard family-specific primers. Synthesis of patient specific primers was based on defined nucleotide sequence of clonal PCR-product. Monitoring MRD was realized by nested PCR using patient specific primers. Results: IgH and/or TCRG genes rearrangements were detected in 50 of 59 patients (84,7%). IgH complete rearrangements were found in 31of 43 patients (72,1%) with B-lineage ALL, IgH incomplete rearrangements were detected in 12 of 43 patient (27,9%) with B-lineage ALL and in one of 15 patient with T-lineage ALL. TCRG rearrangements were found in 13 of 15 patients (86,7%) with T-lineage ALL and in 24 of 43 patients (55,8%) with B-lineage ALL. Seventeen (34%) of 50 patients had one clonal marker. So, phenomenon of oligoclonality was revealed in 33 patients (66%). Two clonal rearrangements were detected in 23 cases, 3 rearrangements were detected in 7 cases and 3 patients had 4 rearrangements. In 3 of 27 patients, in whom all markers were followed, monitoring of MRD showed different pattern of tumor subclones elimination. Monitoring of MRD in patients who had also chromosomal abnormalities such as t(9;22), t(4;11) was carried out by FISH and PCR in Real-time. In one case the patient had 3 IgH rearrangements and chromosomal abnormality t(4;11) at the diagnosis. Later at various stages of chemotherapy we observed a different clearance of IgH rearrangements, however, when cytogenetic remission was achieved and MLL-AF4 expression was absent there were two of three IgH markers. This fact confirms the presence of several tumor subclones, their different clearance during the treatment and suggests persistence of tumor in this patient. Conclusion: In ALL patients with several subclones MRD monitoring should carried out with all markers, as we proved different subclone clearance suggesting different cells sensitivity to cytostatic drugs. Disclosures: No relevant conflicts of interest to declare.
4333 Adult acute lymphoblastic leukemia (ALL) differs from pediatric ALL by higher frequency of unfavorable biological features including cytogenetics (often t(9;22), rare t(12;21)), slower molecular response (MRD negativity is lower at day near +30 in adults - 47% vs 80%; Bruggemmann, Blood, 2006; Borowitz, Blood, 2010), more toxicity followed by less complience, all this translating in less efficacy. Another very important, early and simple predictor of antileukamia effect in ALL is prednisolone (PRD) sensitivity, that is to say tumor clearance within one week of prephase. It's a well documented fact in childhood ALL, but scarcely characterized in adults. 35% of adults with ALL are considered to be resistant to PRDN compaired to 10% children after evaluation of PB blast count on day +8 (Annino, Blood, 2002; Shrappe, Leukemia 2002), but few data exists about bone marrow blasts clearance. We initiated a prospective multicenter trial for Ph-negative ALL under the age of 55 based on: 1.evaluation of blast clearance in b/m after 7 days of PRD and its substitution by dexamethazone (DEXA) if blast count was 25% and more. 2. “no interruptions” protocol with 8 weeks induction and 5 consolidation phases followed by 2-years maintenance. 3. prolonged L-asparaginase application at 10.000 IU weekly in induction, once in two weeks in consolidations, twice a month in maintenance (total proposed dose 560.000 IU). The study started in April, 2009. 20 participating centers enrolled 77 patients (median age 27y (16-55), 44f, 33m, 61,5%=B-lin, 38,6%=T-lin; 41% with normal karyotype (NK)). 30,7% of patients were in the standrad risk (SR) group (WBC <30 for B-Lin, <100 for T-Lin, EGIL BII-III, T-III; LDH < 2N, No late CR, t(4;11)-), 69,3% - in the high risk (HR) group (WBC >30 for B-Lin, >100 for T-Lin, EGIL BI, T-I-II-IV; LDH > 2N, No late CR, t(4;11)+). The analysis was performed in June, 2010, and comprised 70 pts. The data on the day +8 b/m count was reported in 67 pts: 70% of them had b/m blasts 25% and more, thus were considered as non-responders to PRD (60 mg/m2) and were switched to DEXA (10 mg/m2). It's worth to note that the proportion of non-responders to PRD was almost equal in the SR and HR groups: 12 of 20 (60%) in SR and 35 of 47 (74,5%). CR rate was high in both risk groups (SR=95,5%; HR=89,4%) and immunological subsets (B=91,4%;T=91,6%). For the whole group of analysed patients (n=70) there were 5 induction deaths (7,1%) and 1 resistant leukemia (1,4%). Median of days without treatment during induction period was 8 days (0-56). Death in remission was reported in 2 of 64 CR pts (3,1%). Relapses occurred in 4/64 (4,2%). Within the short period of follow-up (14 mo) the probability of OS for 70 patients constituted 78,8%, DFS – 76,7%, continuous CR – 81,2%. The difference in DFS between PRD responders and non-responders was at borderline: 63,3% vs 93,8% (p=0,1), and statistically proved in pts with NK vs all other abnormalities: 100% vs 72% (p=0,03). Age, WBC, immunophenotype, risk group, time without treatment did not influence survival. We concluded that in adult Ph-negative ALL the proportion of non-responders to PRD is very high (70%), thus providing much poorer results than in children; sensitivity to PRD may still be used as very simple discriminative marker of unfavorable prognosis. Disclosures: No relevant conflicts of interest to declare.
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