ALL Pts Research Articles

Bcr-Abl Kinase Domain Mutations in Imatinib and in Second-Generation Tyrosine Kinase Inhibitor Eras: Seven Years of Mutation Analysis, a Report by the GIMEMA CML Working Party

AbstractAbstract 2279Over 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.

Whole-Transcriptome Sequencing In Chronic Myeloid Leukemia Reveals Novel Gene Mutations That May Be Associated with Disease Pathogenesis and Progression

AbstractAbstract 885Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) has always been regarded as a genetically homogeneous disease. However, the fact that a proportion of patients (pts), especially in the high Sokal risk setting, fail tyrosine kinase inhibitor therapy and progress to blast crisis (BC) suggests that a certain degree of heterogeneity exists. It can be hypothesized that genetic factors additional to the Ph+ chromosome may be present in these pts. To address this issue, we are currently using massively parallel sequencing to perform a qualitative and quantitative survey of the whole transcriptome of Ph+ CML cells at diagnosis and at progression to BC. Results are being integrated with genome-wide search for copy number alterations by Affymetrix SNP 6.0 arrays. We used a Solexa Illumina Genome Analyzer to scan the transcriptome of a CML patient at the time of diagnosis, at the time of remission (major molecular response) and at the time of progression from chronic phase (CP) to lymphoid blast crisis (BC). Both custom scripts and published algorithms were used for read alignment against the human reference genome, for single nucleotide variant (SNV) calling, for identification of alternative splicings and fusion transcripts, and for digital gene expression profiling. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs detected in the remission sample – representing inherited sequence variants not specific for the Ph+ clone – allowed the identification of eight missense mutations at diagnosis affecting the coding sequences of AMPD3 (encoding adenosine monophosphate deaminase 3), SUCNR1 (succinate receptor 1), FANCD2 (Fanconi anemia, complementation group D2), INCENP (inner centromere protein), BSPRY (B-box and SPRY domain containing), HEXDC (hexosaminidase containing), NUDT9 (ADP-ribose diphosphatase) and KIAA2018 (encoding a protein with predicted DNA binding and transcriptional regulation activity) genes. Six of these mutations (FANCD2, INCENP, BSPRY, HEXDC, NUDT9) were also detected in the Ph+ clone re-emerged at the time of disease progression, together with seven additional missense mutations affecting the coding sequences of IDH2 (isocitrate dehydrogenase isoform 2), DECR1 (2,4-dienoyl CoA reductase 1), C4Orf14 (mitochondrial nitric oxide synthase), MRM1 (mitochondrial rRNA methyltransferase 1), PRKD2 (protein kinase D2), TCHP (mitostatin) and ABL1 genes. Digital gene expression analysis showed downregulation of SUCNR1, that might be a consequence of the P292A mutation we detected. IDH2, MRM1, AMPD3, and KIAA2018 mutations were found in additional pts. The IDH2 R140Q mutation was detected in 3/75 (4%) myeloid BC, 1/31 (3.2%) lymphoid BC, 0/34 Ph+ ALL and 0/23 Philadelphia-negative (Ph-) ALL pts. The MRM1 C120S mutation was found in 6/70 (9%) additional BC pts (2 lymphoid and 4 myeloid). AMPD3 and KIAA2018 genes were found to harbour the same point mutations (N334S and S1818G, respectively) in 1 out of 20 additional CP patients analyzed. Massively parallel sequencing of the sample collected at diagnosis also revealed that the Bcr-Abl kinase domain was already harbouring point mutations at low levels (E308D, A344G, R386S) but not the T315I that was selected at the time of disease progression. Point mutations in untraslated regions where miRNAs are known to bind were also detected, and are currently under validation. Digital gene expression profiling comparing progression to diagnosis showed significant expression changes including upregulation of 134 genes and downregulation of 88 genes. In particular, we observed an upregulation of the B-cell developmental factor PAX5, its interactor Lef-1 and its targets IRF4, BLNK, Bik, EBF1, CD79A, CD79B, CD19, VpreB1, VpreB3, BOB1, RAG1 and RAG2; upregulation of PAX9; upregulation of WNT3A, WNT9A, GLI3 and downregulation of SFRP1, resulting in aberrant activation of the Wnt signalling pathway. In summary, our preliminary data highlighted putative key genes whose deregulation may be recurrent in a subset of CML patients and may be linked to disease pathogenesis or progression. Their actual role in CML is currently being exlored. Massively parallel sequencing of additional patients is ongoing. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants. Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Treatment of Acute Leukemia During Pregnancy – 20 Years Experience

AbstractAbstract 4348Acute leukemia during pregnancy is a rare but not an unique event: 1 case per 75–100.000 pregnant women and the majority of cases are registered in the 2nd/3rd trimester. The main law has to be carried out in this life threatening situation: to save two lives - mother's and child's. Though in the 1st trimester medical abortion is highly recommended, in the 2nd/3rd trimester chemotherapy should be applied without dose corrections. It is considered to be of no major danger to the fetus and gives good chances to the mother.Here we would like to report our 20 years experience with 32 pregnant women (median age 25y (19-35)) with acute leukemias: 13 AML, 5 APL and 14 ALL. 26 (81%) of them were diagnosed with AL in the 2nd/3rd trimester. We used the following approach: 1. up to 12 weeks of pregnancy medical abortions were performed (6 pts - 1 AML, 1- APL, 4-ALL); 2. at 35–40 weeks of pregnancy “cesar sections” were done and then chemotherapy was started (7 pts – 4 AML, 2 APL, 1 – ALL). All 7 children are alive and well; 3. at 13–34 weeks of pregnancy standard chemotherapy was applied according to AL subtype treatment protocol (19 pts – 8 AML; 2 APL, 9 ALL). In AML 7+3 protocol (ARA-C – 100 mg/m2 bid 1–7 days, Daunorubicin 45 mg/m2 (in 3 pts) or 60 mg/m2 1–3 days), in APL – 7+3+ATRA or AIDA (2 pts), in ALL - 8 weeks induction and prolonged post-CR therapy - were used.All together in 13 AML pts there were 9 CR (69%), 2 ED (15,5%), 2 DR (15,5%); in 5 APL pts – 4 CR (80%), 1 ED (20%); in 14 ALL pts – 9 CR (64,3%), 3 DR (21,4%), 2 ED (14,3%). 85% of pts had infectious complications during induction, 1 ATRA-syndrome, 1 emergency “cezar section” was performed at 30th week of pregnancy due to premature placenta unlayment (PPU) with hemorrhage due to L-asparaginase. There was one late spontaneous abortion (+21 weeks), no deaths and no defects were registed among newborns “treated” in uteri (n=18), all of them were followed in micropediatriac departments, neutropenias was registed in half of them and pneumonias in 3 (17%). All children (n=25) are alive and well. The oldest is 20 years old now, the youngest – 10 months. The probability of 5-years overall survival in AML pts was 34,6%, in ALL – 26,7%. In APL only 1 pt is alive in 1st CR due to 1 death in consolidation and two relapses.Within the period of the study we have developed some practical recommendations: 1. Daunorubicin in 7+3 courses should be used at 60 mg/m2 as there were no CR in pts receiving 45 mg/m2. It's a crucial point as CR must be achieved after the 1st course, especially in 30–32 weeks of pregnancy because delivery must be carried out in stable status. 2. Idarubicin can be used safely in resistant to daunorubicin AML pts and in AIDA protocol for APL. AIDA is less toxic than 7+3+ATRA for APL induction and well effective. 5-days mitoxantrone consolidation should be postponed to 3–4 months after delivery. 3. L-asparaginase should not be applied in ALL treatment during pregnancy (only after delivery) due to coagulation disturbances and possibility of premature placenta unlayment (PPU). 4. Delivery during AL treatment must be planned at 34–36 weeks of pregnancy. 60% of our patients had “cesar sections”. 5. Every day gynecologists care is absolutely needed (uteri hypertonus, fetus hypotrophy, PPU, etc). 6. Chemotherapy restart should be planned 3–4 weeks after delivery because immediate (within 5–7 days) continuation of cytostatic treatment caused severe combined infections complications due to postdelivery immunodeficiency and desadaptation. In case of resistant leukemia restart treatment in 2 weeks and not with high-dose protocols. 7. In newborn children all complications can be cured within 1–5 weeks in special micropediatic departments, children grew up healthy and intelligent. 8. The results in ALL pts with pregnancy seem to be worse than in general ALL population. 9. After CR in APL careful monitoring of MRD may provide better outcome and avoid aggressive consolidation courses.The main conclusion that comes up from this data is the obvious necessity to treat a pregnant woman with acute leukemia diagnosed in the 2nd/3rd trimester with adequate chemotherapy, that results in saving the child's life and - in many cases – the mother's. The overall survival in pregnant women with acute leukemia is quite similar to the outcome in all patients, though we wished it to be better. Disclosures:No relevant conflicts of interest to declare.

Phase I trial of AP24534 in patients with refractory chronic myeloid leukemia (CML) and hematologic malignancies.

6511 Background: AP24534, an oral multiple tyrosine kinase inhibitor (TKI), is a potent pan-BCR-ABL inhibitor, including T315I. A phase I trial to assess its safety, maximum tolerated dose and activity is ongoing. Methods: Patients (pts) with refractory leukemia were enrolled on a dose escalation trial of daily AP24534. Dose levels were 2, 4, 8, 15, 30 and 60mg and a 45mg level is currently open. Results: As of 1/4/10, 48 pts, median age 62 yrs (range 26-85) have been enrolled. There are 42 CML pts (31 chronic [CP], 6 accelerated [AP], 5 blast phase [BP]) and 2 with Ph+ ALL. Prior therapies in CML pts include imatinib (I) (100% of pts), dasatinib (D) (88%), nilotinib (N) (65%); 97% and 81% of the Ph+ pts failed 2 and 3 prior TKIs, respectively and 88% had documented BCR-ABL mutations. Ph+ pts include 18 with T315I and 6 with F317L. 32 (67%) remain on therapy, median duration 61 days (range 5-360). Sixteen (33%) have discontinued; 7 (15%) for PD, 6 (13%) for investigator decision, 2 (4%) for unrelated deaths, and 1 (2%) for toxicity (pancreatitis). Related AEs ≥ 10% are: nausea (20%), fatigue (15%), vomiting (15%), headache (13%), arthralgia (10%), hot flush (10%), incr glucose (10%), incr lipase (10%), muscle spasms (10%), and rash (10%). Grade 3 or 4 thrombocytopenia occurred in 40% pts and neutropenia in 43%. There are 4/12 pts with G3/4 amylase/lipase at 60 mg and 2/12 with G2 reversible pancreatitis. In evaluable CML pts, 11/13 (85%) achieved CHR, 7/21 (33%) achieved CCyR and 10/21 (48%) achieved MCyR. All 7 (100%) evaluable T315I CP CML pts had CHR, 4 (57%) had CCyR, and 1 (14%) MiCyR. Of 11 evaluable AP/ BP CML or Ph+ALL pts, 4 (36%) had MHR, 1 (9%) had CCyR, and 1 (9%) had minimal response. Of 8 T315I pts in this group, 3 (38%) had MHR, 1 (13%) had CCyR, and 1 (13%) had minimal response. There was 1 CCyR and 1 minimal CyR in 2 F317L pts who failed I, D, and N. A F359C pt who failed I and N achieved CHR and CCyR. MMR was achieved in 3 CML pts (M351T, F359C, and F317L) after 3 months (2 at 15 mg and 1 at 60 mg) and 1 T315I pt attained a transcript level of 0.15% after 6 months on 30mg. Conclusions: AP24534 is well tolerated with no DLTs until 60 mg. There is evidence of antileukemia activity in pts resistant to second generation TKIs, including pts with T315I. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ARIAD Pharmaceuticals Novartis ARIAD Pharmaceuticals ARIAD Pharmaceuticals

Prevalence of familial malignancy in a prospectively screened cohort of patients with leukemia.

6590 Background: Newer studies are giving increased understanding of the genetic origins of cancer. We conducted a pilot study to determine the prevalence of familial malignancy among adult leukemia patients admitted to a leukemia service from April 2009 to Dec 2009. Methods: An IRB approved screening questionnaire was used to ascertain information from 104 leukemia patients. Pts who were adopted or uncertain of their family history were excluded. Fisher's exact test for analyzing categorical variables and a two-sample t-test for continuous variables were used. Results: Of the 92 pts analyzed, 41 (44.6%) reported a first degree relative and 61 (66.3%) reported a first or second degree relative with cancer. At least one first degree relative with cancer was reported from 66.7% of CLL pts, 54.9% of AML pts, 37.5% of ALL pts, and 27.3% of CML pts. The median age was 55 yrs for those pts with either a first or second degree relative with cancer and 62 for those without a family history (p = ns). The most common cancers reported were breast (20% of pts reported an effected relative), colon (13%), lung (12%), leukemia (9%), unknown cancer (8%), prostate (6%) and stomach (5%). In many cases, the percentage of family members with a specific cancer type was similar to that reported for the general US population (Jemal et al, Cancer J Clin 2009; 59:225- 249). However, significant differences were noted when comparing our pts' family history with that of the US population for the following cancer subtypes: leukemia (9% our pts vs. 3% US), stomach (5% vs. 1%), colon (13% vs. 7%) and NHL (0% vs. 4%). Numerical differences that were not significant were noted for breast (20% vs. 13%), prostate (6% vs. 3%), bladder (1% vs. 5%) and esophagus (3% vs. 1%). Nine pts had 12 family members with leukemia. The median and mean age of these pts was 50 and 47 resp compared with the remaining pts who had a median age of 59 and mean of 57 (p = 0.04). Of these pts, 5 had AML, 3 had CML and 1 had ALL. Eight pts had a Li-Fraumeni pattern of familial clustering of cancer. Conclusions: The pattern of familial cancers may be different for pts with leukemia compared to the general population. We continue to accrue pts to determine if these differences effect treatment outcome. No significant financial relationships to disclose.

A Phase 1 Trial of Oral AP24534 in Patients with Refractory Chronic Myeloid Leukemia and Other Hematologic Malignancies: First Results of Safety and Clinical Activity against T315I and Resistant Mutations.

A Phase 1 Trial of Oral AP24534 in Patients with Refractory Chronic Myeloid Leukemia and Other Hematologic Malignancies: First Results of Safety and Clinical Activity against T315I and Resistant Mutations.

Alemtuzumab can be Incorporated Into Front-Line Therapy of Adult Acute Lymphoblastic Leukemia (ALL): Final Phase I Results of a Cancer and Leukemia Group B Study (CALGB 10102).

Abstract 838Eradication of minimal residual disease (MRD) during the first months of ALL treatment is associated with improved disease-free survival (DFS). We hypothesized that alemtuzumab, a humanized anti-CD52 monoclonal antibody, might be an effective, novel agent for eradication of MRD. In CALGB 10102, we tested dose escalation of alemtuzumab in sequential cohorts of CD52+ cases to a target dose of 30 mg administered SC 3 times/week for 4 weeks (12 doses) during post-remission therapy. Dose limiting toxicity (DLT) was defined as the inability to proceed with protocol treatment within 6 weeks after the last dose of alemtuzumab. Eligible patients (pts) had ≥10% lymphoblast CD52 expression at diagnosis determined in a CALGB reference laboratory. Antimicrobial prophylaxis for cytomegalovirus (CMV) and Pneumocystis carinii was mandated. The 10102 therapy consisted of 6 monthly chemotherapy modules followed by maintenance therapy for a total of 2 years (Stock et al. ASH 2005, abstr 145); 299 untreated ALL pts were eligible and enrolled from 2003–2007. We previously reported (Lozanski et al. ASH 2007, abstr 2386) that 70% were CD52+ and eligible to receive Alemtuzumab (70% of B-cell ALL; 53% of T-cell ALL; 100% of Ph+ cases). Results:Twenty-four pts in remission (CR1) received Alemtuzumab as their fourth treatment module during the Phase I portion of the study. The median age was 37 years (18-77 years); 18 (80%) had B-cell ALL; 5 (19%) had T-cell ALL. 17/24 cases had evaluable cytogenetics after central review: 5 had favorable, 8 intermediate-risk, 2 poor-risk cytogenetics (neither were Ph+), and 2 were not classifiable (miscellaneous abnormalities). Non-hematologic toxicities were mild. SC alemtuzumab was well tolerated. Grade 3-4 myelosuppression was reported during 4 weeks of alemtuzumab treatment in 4 pts; 2 pts had Grade 3-4 lymphopenia. 4 pts had DLT: subsequent treatment was delayed in 2 due to transient CMV viremia; 1 developed Staph aureus empyema; and 1 had prolonged myelosuppression but did not receive G-CSF support as recommended by the protocol. 3 pts relapsed with ALL immediately following completion of the alemtuzumab module. Serial assessment of MRD using quantitative clone-specific PCR was possible in 11/24 cases. There was a median 1-log decrease in MRD during alemtuzumab therapy in the 20 and 30 mg cohorts. However, there was a 2-log rise during alemtuzumab therapy in one pt who relapsed 6 weeks later. Pharmacokinetic analysis revealed rising alemtuzumab serum levels during treatment in all dose cohorts, and levels were still detectable in some pts 10 weeks after completing alemtuzumab. During subsequent post-remission therapy, 8 pts developed CMV viremia, 2 had Herpes simplex infections, and 3 had Herpes zoster reactivation. There was not a significant correlation between serum alemtuzumab level and change in MRD or risk of viral infection. The median follow-up time for the 14 surviving pts is 51 months (49-54 months). Median DFS is 53 months (95% CI, 39 – not reached); median overall survival is 55 months (95% CI, 42- not reached). Conclusions:CD52 is expressed in the majority of adult ALL cases. Addition of alemtuzumab to front-line therapy is feasible and provides reduction in MRD, but is associated with viral infections. Dose escalation of alemtuzmab was not associated with DLT. Based on these results, the 30 mg dose of alemtuzumab was explored in an additional 70 patients in the Phase II portion of the study. The encouraging DFS results reported here require confirmation from longer follow-up of the larger Phase II cohort of patients. Disclosures:Stock:Genzyme: Research Funding. Off Label Use: The testing of alemtuzumab for treatment of ALL in adults. Lozanski:Genzyme: Research Funding. Vij:CELGENE: Honoraria, Research Funding, Speakers Bureau. Powell:Antisoma: Research Funding. Sher:invivoscribe: Employment. Richards:Genzyme: Employment. Sung:genzyme: Employment.

Rapid Engraftment of Neutrophils and Platelets with Mesenchymal Stem Cell Based Cord Blood (CB) Expansion (EXP).

Abstract 3394Poster Board III-282CB transplantation (CBT) is often complicated by delayed or failed engraftment. We conducted a study of ex vivo co-culture of CB mononuclear cells with either third party family member ≥ 2/6 HLA matched marrow derived MSCs (N=8) or off-the-shelf mesenchymal progenitor cells (MPCs) from Angioblast Systems Ltd. (N=9). MSCs provide a microenvironment for the generation of complex molecular cues that direct proliferation and regulate the differentiation and maturation of hematopoietic progeny. Patients had two CB units matched in at least 4/6 HLA antigens, with a minimum of 1×107 TNC/Kg per unit. Methods:Diagnoses were AML/MDS (N=10), ALL (N=3), NHL (n=1), MM (n=1), and CLL (N=2). Five patients (29%) were in CR (CR1, n=1) and 12 (71%) had active disease at CBT. Preparative regimen: myeloablative fludarabine, melphalan, thiotepa and ATG (n=17), with rituximab in the 3 ALL pts. GVHD prophylaxis: tacrolimus and MMF. Median weight was 79.7 Kg (range, 15-102) and median age was 36 years (2-55 years). Donor-recipient HLA matching was 5 of 6 in 29% of the cases and 4 of 6 in 71%.Ex-vivo EXP: 100 ml of marrow was aspirated from the family donor and MSCs generated in ten T175 flasks, which took ∼21 days (n=8) or one vial of Angioblast MPCs was thawed and expanded to confluence in 10 flasks within 4 days (n=9). The CB unit with the lowest TNC dose was then thawed, divided into 10 fractions, and each placed into 1 flask containing the confluent layers of MSCs/MPCs in expansion media with SCF, FLT3, G-CSF and TPO. After 7 days at 37°C, the non-adherent cells were removed from each flask, placed into each of ten one-liter Teflon-coated culture bags (American Fluoroseal) and cultured for an additional 7 days (14 days total), while 50 ml of media/growth factors was added to the flasks to culture the remaining adherent layer during that time period. On day 14 the cells from the bags and the flasks were combined, washed and infused along with a second unmanipulated CB unit. Results:The median number of infused total nucleated cell (TNC) and CD34+ cells per kg in unmanipulated CB was 2.3 × 107 (range, 1.9-8.2) and 1.6 × 105 (range, 0.3-1.26). There were no toxicities attributable to the EXP cells. The median TNC fold-EXP for recipients of family-MSCs was 11.7 (0.7-28) and for Angioblast-MPCs was 15.5 (3.1-22.5); Fold-EXP of CD34+ cells was 11.7 (1.7-50.1) for family-MSCs and 46.6 (3.85-71.9) for Angioblast-MPCs. The median number of infused TNC and CD34+ cells per kg after EXP for all pts was 5.8 × 107 (range, 0.1-1.43) and 6.05 × 105 (range, 0.18-30). Median time to neutrophil and platelet engraftment was 15 days (9-25 days) and 37.5 days (13-56). Sixteen (94%) and 14 (82%) of all patients engrafted neutrophils and platelets, respectively. One patient died before engraftment. All evaluable patients became complete donor(s) chimeras. One CB unit dominated in all patients; on transplant day +21, EXP unit contributed with a mean of 10% of T cell and 24% of myeloid chimerism; on day +40, corresponding proportions were 2% and 4%, and on day +70, 0% and 3%, respectively. Acute grade II-IV and III-IV GVHD rate was 38% and 12%, while 50% of at-risk patients developed chronic GVHD. Ten patients (59%) are alive (3-14 months after CBT), while 7 patients have died due to infections (n=4), relapse (n=2) and GVHD (n=1). Actuarial 6 and 12 month survival is 70% and 50%, respectively. Conclusion:The decreased logistical demands and expansion results demonstrate that off-the-shelf Angioblast-MPCs are the preferred stroma for this CB EXP procedure. MSC/MPC-CB EXP is feasible and may provide rapid engraftment of neutrophils and platelets. Platelet engraftment occurred in a high proportion of patients (80%) in this cohort of high-risk patients with a median weight of 80 Kg. Disclosures:No relevant conflicts of interest to declare.

Imatinib and Interferon-Alpha Maintenance Therapy for Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ALL) Ineligible for Allogeneic Stem Cell Transplantation (SCT).

Abstract 2042Poster Board II-19 Introduction:The tyrosine kinase inhibitor imatinib (IM), alone or in combination with chemotherapy, has become the mainstay of front-line treatment for Ph+ ALL. We recently demonstrated that IM in combination with intensive consolidation chemotherapy of approximately one year duration is feasible in elderly patients with de novo Ph+ ALL, but is associated with a high relapse rate. Allogeneic SCT (alloSCT) is potentially curative but may not be feasible in most elderly or comorbid patients. Maintenance therapy (MT) in such patients is conceptually attractive, but data on whether any type of MT facilitates long-term leukemia-free survival of patients with Ph+ALL in first CR is lacking. Based on data suggesting that interferon-alpha (IFN-a) possesses anti-leukemic activity in patients with Ph+ALL, we conducted a phase II study to determine the feasibility and efficacy of MT consisting of imatinib in combination with low-dose (LD) IFN-a in elderly patients with Ph+ALL who were not eligible for SCT. In addition, we examined whether bcr-abl transcript levels and mutation status were predictive of relapse and remission duration. Methods:Nineteen elderly patients (median age 66 yrs; [60-75 yrs.]) who had received IM-based remission induction and consolidation therapy as reported previously (Ottmann et al., Cancer 109:2068-76, 2007) were enrolled in a clinical trial of IM 400 mg daily in combination with LD-IFNa with a target dose of 3 Mio IU/week. At the time of enrollment, the majority of patients (n=12) had received five (n=3) or six (n=9) consolidation cycles, the remaining patients had discontinued intensive front-line therapy after four (n=2), three (n=1), two (n=1), and one (n=1) consolidation cycles, 2 patients were switched to MT after induction. The median number of cycles of consolidation chemotherapy given concurrently with IM was six. Minimal residual disease (MRD) was serially assessed by quantitative RT-PCR, mutational analyses was performed by D-HPLC and direct sequencing. Results:The median overall duration of MT is 26 mos. (range 3-92 mos.). Seven of 19 pts. (37 %) are in ongoing CR, with a median remission duration from start of maintenance of 76.7 mos. (54-91 mos.). Median overall survival of all pts. is 61 mos. (range: 20-99 mos.). Eleven of 18 evaluable pts. experienced side effects which lead to a dose reduction of IFN. 9 pts. suffered from moderate depressions or fatigue. Hematologic toxicity was mild: only 2 pts. developed grade 3 cytopenia during MT. Remission duration was independent of the number of previous cycles of intensive chemotherapy, of the MRD response during the first 6 mos. of intensive front-line therapy (16 mos. vs. 26 mos.) and of achievement of PCR negativity at any time during intial therapy. Surprisingly, the bcr-abl transcript level at the start of MT likewise had no impact on time to disease recurrence. Conclusions:In elderly Ph+ALL pts. ineligible for allogeneic SCT, maintenance with imatinib in combination with low-dose IFN-a results in long-term sustained remissions in a substantial proportion of patients, with acceptable side effects. Surprisingly, the MRD response and mutation status prior to MT were not significantly predictive of remission duration. Likewise, greater intensity of prior chemotherapy as determined by the number of administered consolidation cycles had no significant benefit with respect to remission duration during MT. More extensive evaluation of tyrosine kinase inhibitors in combination with LD IFNa as MT for Ph+ALL is warrented. Disclosures:Ottmann:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria.

Conditioning Regimens Prior to Allogeneic Stem-Cell Transplantation (SCT) In Acute Leukemia: The Role Of Dose Intensity

Allogeneic SCT with both myeloablative and reduced-intensity conditioning (RIC) is effective therapy in acute leukemia. To better define the role of dose intensity in SCT, we retrospectively analyzed SCT outcomes in 356 consecutive adult patients (pts) with MDS/AML (n = 277) and ALL (n = 79) given SCT over an 8-year period in a single institution. The median age was 51 (range, 17–75). The donors were HLA-matched siblings (n = 191), matched unrelated (n = 139) and alternative (n = 26). Pts meeting standard eligibility criteria were routinely given myeloablative conditioning (MAC, Cy/TBI or BuCy, n = 141). Pts non-eligible for MAC were given either RIC (fludarabine and reduced doses of busulfan or melphalan, n = 116) or modified myeloablative conditioning (modMAC, fludarabine with myeloablative doses of busulfan or treosulfan, n = 99). Disease status at SCT was CR1/ CR2 (n = 176), previously untreated or untreated relapse (n = 63) and chemo-refractory (n = 117). With a median follow-up of 30 months (range 1–103), 159 pts are alive and 197 died; 75 of treatment-related causes and 122 of relapse. The estimated 5-yr overall survival (OS) in this relatively high-risk pt group was 34% (95%CI, 27–41). The status of disease at SCT was the most important factor predicting OS; 48, 31 and 16% for pts in CR, untreated or chemo-refractory leukemia, respectively (p<0.001). Multivariable analysis (MVA) identified SCT not in CR, SCT from unrelated and alternative donors as adverse prognostic signs with hazard ratios (HR) of 2.7, 1.5, and 2.2, respectively. The conditioning regimen used was not a significant factor in the whole pt group with 5-yr OS of 38, 35 and 31% after MAC, modMAC and RIC, respectively (p = NS). However, RIC was associated with reduced OS in 2 subgroups. Among pts with MDS/AML given SCT not in CR, 5-yr OS was 34, 24 and 13% after MAC, modMAC and RIC, respectively (p = 0.03) with HR of 1.6 for RIC in MVA. In the group of ALL pts, MVA identified RIC and SCT not in CR1 as adverse prognostic signs with HR of 2.0 (p = 0.04) and 2.0 (p = 0.03), respectively. In conclusion, MAC should still be considered the standard of care for SCT in acute leukemia in eligible pts. RIC is associated with inferior outcome in pts with ALL and pts not in CR at SCT. Randomized studies are needed to determine the role of RIC in MDS/AML pts in CR at SCT. The novel modMAC regimens are relatively well tolerated even in pts not eligible for MAC and may be more effective than standard RIC in refractory disease.

Dexamethasone(DEX)(6mg/sm/d) and Prednisolone(PRED)(60mg/sm/d) in Induction Therapy of Childhood ALL Are Equally Effective: Results of the 2nd Interim Analysis of EORTC Trial 58951

Background: there is some biologic and clinical evidence that DEX is more efficient than PRED at similarly anti-inflammatory doses in the treatment of childhood ALL. Previous trials have called for PRED doses (40 mg/sm/d) lower than those of PRED traditionally used in EORTC trials. In order to check whether the claimed superiority of DEX might be dose-dependent, we conducted a randomized phase III trial comparing DEX (6 mg/sm/d) to PRED (60 mg/sm/d) (conventional dose) in induction therapy.Methods: the trial was opened in 12/1998 to all newly diagnosed children with ALL up to 18 y. of age. After the opening of the Interfant trial in 1999, children <1 y. old were registered in the latter. Ph1 pts recruited in this 58951 trial were kept in the present analysis because the switch to the EsPhALL was only after induction. The treatment regimen was of BFM type, without CNS radiation even in case of initial CNS leukemia. Randomization was done either on day 1 of the prephase (whenever feasible) or on day 8. In the latter case, PRED was administered before day 8. Non-very high risk pts were further randomized to prolong versus conventional duration of L-asparaginase courses during consolidation and late intensification. Part of average risk pts were also randomized (N=384) for the addition of pulses of VCR + glucocorticoid (same as that assigned for induction) in continuation therapy. Randomization was done centrally, stratification factors being disease, WBC, age, sex, timing of randomization and center. The main endpoint was EFS; secondary endpoints were OS and toxicity. Two interim analyses were foreseen in the protocol. Intent-to-treat analysis was used.Results: between 12/1998 and 2/2008, a total of 1853 ALL pts were randomized; as of 2/2008, 1703 pts were evaluable for EFS analysis. At a median follow up of 4.3 y., the 5 y. EFS rate was 82.1% (SE 1.5%) for the pts in the DEX arm and 82.4% (SE 1.5%) in the PRED arm (hazard ratio (HR) = 0.99, 98.8% CI 0.72–1.36, p=0.94); the futility boundary was crossed. In precursor-B ALL pts, the 5 y. EFS rate was 84.2% (DEX) vs 84.1% (PRED) (HR=0.95, p=0.75) and in precursor-T ALL the 5y. EFS rate was 70.7% vs 72.0% (HR=1.06, p=0.83). Isolated and combined CNS relapse crude rates were 1.1% each (DEX) vs 1.6% and 2.2% (PRED) respectively (resp.). The 5-y CNS incidence rate was 2.9% (DEX) vs 4.9% (PRED), the non-CNS incidence rate was 10.9% (DEX) vs 9.4% (PRED), and the 5-yr death in CR incidence rate was 2.7% (DEX) vs 2.1% (PRED). During induction the incidence of grade 3–4 infection was very similar in the two arms. During post-induction consolidation (“protocol IB”), it was higher in the DEX arm (23.5% vs 21.1%), in particular in pts randomized to prolonged L-asparaginase arm: 29.1% (DEX) vs 21.8% (PRED).Conclusion: DEX and PRED, used at the doses of resp. 6 and 60 mg/sm/d during induction, had equal efficacy. DEX decreased by 2.0% the 5-yr CNS incidence rate but increased by 1.5% the non-CNS incidence rate and by 0.6% the death in CR incidence rate. DEX in induction entails a higher infection rate during the consolidation, particularly when L-asparaginase is pursued throughout this phase. The optimal dosage of either glucocorticoid, compatible with acceptable toxicity, remains to be determined.

Implications for the Use of Monoclonal Antibodies in Future Adult ALL Trials: Analysis of Antigen Expression in 505 B-Lineage (B-Lin) ALL Patients (pts) on the MRC UKALLXII/ECOG2993 Intergroup Trial.

MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG's reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p<0.0001). While CD65(s) and CD15(s) did not affect outcome, the combined expression of CD33/ CD13 conferred an inferior prognosis; this effect was due to the association of CD33/ CD13 with BCR/ABL positivity (p<0.0001). BCR/ABL negative B-Lin ALL (N=350) expressed CD33 on a median of only 4% of blasts, compared with a median of 54% in BCR/ABL positive ALL (N=152) (p=3.5E-09). However, intensity of CD33 staining was significantly lower in both BCR/ABL negative and positive blasts (median 3.1) compared with leukemic myeloblasts (median 56) (p=<0.001). Median CD52 expression was higher in BCR/ABL positive versus negative B-Lin ALL (p=3.5E-06); in neither group was CD52 associated with outcome. We conclude that antibodies to CD52 are better suited for BCR/ ABL positive than negative B-Lin ALL. Since expression of CD33 in ALL is weak, CD33- independent effects of gemtuzumab must be investigated. Low CD20 combined with high CD22 expression confers superior OS. The expression of these Ags in most B-Lin ALL pts justifies the incorporation of antibodies into frontline chemotherapy regimens.B-Lin ALL StageCD20 PercentageMedian IntensityCD22 PercentageMedian Intensity≥20%≥50%≥75%≥20%≥50%≥75%Pro-B21103288725512Early PreB6648372196908428Pre-B61463721100908137Mature B8780641069284809

Minimal Residual Disease and Mutational Status Prior to and after SCT for Patients with Philadelphia-Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

BCR-ABL Tyrosine Kinase Domain (TKD) mutations have been identified as a major mechanism of acquired resistance in the majority of Ph+ ALL patients who relapse during treatment with ABL kinase inhibitors such as imatinib (IM). TKD mutations are detectable with low frequency in 30%–40% of elderly, IM-naïve patients with newly diagnosed, Ph+ALL. The proportion of younger Ph+ALL patients who already harbor a TKD mutation prior to IM therapy and the frequency with which these mutations develop during combined treatment with IM and intensive chemotherapy are not known. Moreover, the impact of allogeneic SCT on different types of TKD mutations and its ability to eradicate mutations in patients transplanted in CR1 as opposed to CR2 remain to be established. It was the aim of this analysis to determine the impact of combination therapy with imatinib and chemotherapy followed by allogeneic SCT on MRD levels and the frequency of BCR-ABL mutations in patients with Ph+ALL undergoing SCT as front-line (in CR1) as opposed to second-line (≥ CR2) treatment. Bone marrow (BM) and/or peripheral blood (PB) samples were collected pre-treatment (n=46), during IM-based combination therapy prior to SCT (n=228) and serially after SCT (n=665) from a total of 53 pts. with newly diagnosed Ph+ALL (median age: 45 yrs.) who were enrolled in prospective GMALL trials of IM followed by SCT. Additionally, we examined a total of 536 samples from 27 Ph+ALL pts. (median age: 48 yrs.) who were enrolled in the early phase II trials of IM as salvage therapy and transplanted in PR, CR2 or with progressive disease. MRD and mutational status were analysed by quantitative rtPCR of bcr-abl transcripts and by denaturing high-performance liquid chromatography (D-HPLC) plus cDNA sequencing, respectively. PCR negativity was always confirmed by nested rtPCR. Patients in whom BCR-ABL transcripts were undetectable by both quantitative and nested rtPCR were considered to not harbor a TKD mutation at the time of analysis. In this cohort of younger patients, TKD mutations were detected prior to IM administration in 14 of 47 evaluable patients (29%) with newly-diagnosed Ph+ALL and in 6 of 20 (30%) patients relapsing after chemotherapy. Of the 46 pts. with de novo Ph+ALL who were evaluable within 4 weeks prior to allogeneic SCT, 22 pts. were MRD negative and 24 MRD pos., 8 (33%) of whom had a detectable mutation. Within 6 months after SCT, 41 of 53 transplanted patients had become MRD negative (77%). In 5 of the 16 patients (31%) who displayed a TKD mutation at any time prior to SCT, the mutation was still detectable after alloPBSCT. 3/5 pts. relapsed with the T315I and 4 pts. remained MRD-positive with mutated BCR-ABL. Of the 34 pts. with unmutated BCR-ABL prior to SCT, only 2 pts. displayed a TKD mutation after SCT. One of these converted to PCR negativity, the other patient relapsed with the T315I. In contrast, only 1 of 20 evaluable patients who subsequently underwent SCT as part of their salvage treatment became MRD negative before SCT. Ten of the 19 pts. in whom BCR-ABL transcripts were detectable carried a TKD mutation. In 5 of these 10 pts. with an initial TKD mutation the analysis of MRD and mutational status was possible after alloSCT. 3 pts. converted to MRD-negativity, 2 relapsed with mutated BCR-ABL. BCR-ABL mutations are detected prior to SCT in a clinically significant proportion (30%) of younger adult patients with Ph+ALL. Allogeneic SCT results in long-term elimination of mutant clones in the majority of patients. However, patients remain at risk of relapsing with previously undetectable TKD mutations, some of whom are considered responsive to second generation ABL TK inhibitors.

More Rapid Outgrowth Kinetics of BCR-ABL Mutations in Advanced Versus Newly Diagnosed Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ ALL): Clinical-Translational Evidence for a Contributory Role of Non-Mutational Resistance Mechanisms.

Background: In Ph+ALL, responsiveness to single-agent imatinib (IM) is determined by the disease stage: whereas more than 90% of pts. with newly diagnosed Ph+ALL achieve a complete remission (CR), the CR rate in patients who failed previous chemotherapy is only 20–30%, with a median time to progression of only 2.2 months. Approximately 80% of pts. with Ph+ALL who relapse during IM-based therapy express a BCR-ABL Tyrosine Kinase Domain (TKD) mutation known to confer high-level IM resistance. We recently showed that in 30– 40% of pts. with de novo Ph+ALL, the same mutation found at relapse is already detectable at a low level prior to first IM exposure; in conjunction with the high response rate, this finding indicates limited growth kinetics of the mutant BCR-ABL clones. To elucidate the reasons for the low CR rate observed in advanced Ph+ALL receiving IM salvage therapy, we compared the frequency and type of up-front mutations, the level of mutant clones, and the outgrowth kinetics of mutations during the first 4 weeks of IM monotherapy in de novo Ph+ALL pts. and in pts. who had failed prior chemotherapy.Patients and methods: We employed denaturing high-performance liquid chromatography (D-HPLC) and cDNA sequencing to examine bone marrow and/or peripheral blood samples collected pre-treatment, during therapy and at the time of relapse from 54 pts. with newly diagnosed Ph+ALL (>55 yrs.) enrolled in a GMALL study of combined IM and chemotherapy (n=48) or treated analogously (n=6) and from 67 Ph+ALL pts. who were enrolled in the initial phase II studies of single-agent IM as salvage treatment after prior chemotherapy.Results: Prior to first IM exposure, TKD mutations were detected in 33% (21/63) of pts. with advanced Ph+ALL and 31% (9/29) of pts. with de novo Ph+ALL. The incidence of mutations observed at relapse was substantially higher but did not differ significantly between the two cohorts: 70% in pts. with advanced disease who relapsed on IM monotherapy (33 of 50 pts.; P-loop 48%, T315I 16%, A-loop 2%) and 84% in de novo ALL pts. (n=26), treated with combination therapy (P-loop 46%, T315I 15%, A-loop 9,5%). In contrast, the outgrowth of cells expressing mutated bcr-abl during the first four weeks of IM therapy was considerably more rapid in Ph+ALL pts who had failed prior chemotherapy: mutations were detected after 2 and/or 4 weeks IM in only 44% of patients (8/18 evaluable) with newly diagnosed Ph+ALL, whereas 69% of patients (27/39) with advanced Ph+ALL expressed mutant BCR-ABL on at least one of these timepoints. Moreover, the percentage of mutated BCR-ABL was below 1% in all 8 pts. with a mutation in the former cohort (de novo Ph+ALL), but higher than 5% (range: 5 – 100) in 16 of the 27 advanced disease pts (59%) who had a TKD mutation.Conclusions: In Ph+ALL, the incidence, distribution pattern and level of TKD mutations detected prior to - or at relapse on - IM does not differ between pts with newly diagnosed leukemia and those having failed prior chemotherapy. In contrast, patients with advanced disease are characterised by considerably more rapid outgrowth kinetics of leukemic cells expressing BCR-ABL mutations, providing clinical-translational evidence for a cooperative effect between mutational and non-mutational resistance mechanisms.

Venothromboembolism (VTE) in Patients (pts) with Acute Lymphocytic Leukemia (ALL), Burkitt's Leukemia/Lymphoma (BL) or Lymphoblastic Lymphoma (LL).

It has been reported that cancer increases the risk of VTE 4-6-fold. The incidence of VTE in cancer pts has been estimated at 1 in 200 per year and has been well documented in solid tumors. Far less is known about the incidence of VTE in pts with hematological malignancies although a recent publication (Blom et al, JAMA 293:715, 2005) suggested that pts with hematological malignancies such as lymphoma and multiple myeloma may be at a higher risk of developing VTE than pts with solid tumors. The incidence and risk of VTE has not been well studied in acute leukemias, a population in which prophylaxis is underutilized given the thrombocytopenia associated with intensive chemotherapy. To evaluate the incidence of VTE in pts with hematological malignancies further and to assess the need for VTE prophylaxis, we conducted a retrospective chart review of 299 pts with ALL, BL, or LL who were seen at M.D. Anderson Cancer Center Center from November 1999 to May 2005. Pts received a hyper-CVAD based regimen (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with methotrexate and high-dose cytarabine). The median observation period was 188 weeks (range 1–328 wks) and included post-consolidation chemotherapy, allogeneic stem cell transplant, and/or salvage chemotherapy. Among 298 evaluable pts, 52 (17%) had confirmed VTE by imaging studies. In 7 pts, VTE was the presenting sign of the malignancy. The median age of pts who developed VTE was significantly higher than of those who did not develop VTE, 48.5 yrs (range 19–75) vs. 42 yrs (range 15–83), respectively (p=0.04). With each year increase in age, the risk of having VTE increased approximately by 1.7% (p=0.059). ALL pts with Philadelphia chromosome (Ph) had a higher incidence of VTE than all other pts (p=0.02), and were 1.8 times more likely to have VTE than non-Ph ALL pts (p=0.007). Caucasian pts or those with history of prior VTE had a significantly higher incidence of VTE (p=0.03 and p=0.002, respectively). The risk was 2 fold higher for Caucasians and 12 fold higher for those with a prior history of VTE. At the time of VTE, platelet counts were below 50 x 109/L in 33%, 50–100 x 109/L in 10%, and greater than 100 x 109/L in 57%. VTE occurs in a significant proportion of pts with ALL, BL, and LL. Thrombocytopenia does not preclude the development of VTE. Older age, Ph positivity, race, and history of prior VTE were significantly associated with the development of VTE. Further analysis of other known risk factors such as the use of erythropoiesis-stimulating agents, hormonal therapy, and other comorbidities is underway to better refine the subpopulation at risk.

Nilotinib in Patients (pts) with Relapsed/Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Who Are Resistant or Intolerant to Imatinib.

Background: Ph+ ALL occurs in about 15% of adults with ALL and is characterized by a poor prognosis. Imatinib in combination with systemic chemotherapy has become the standard therapy for adults with Ph+ ALL. Nilotinib is a highly specific BCR-ABL inhibitor, which is 30-fold more potent than imatinib and active in vitro against 32 of a panel of 33 BCR-ABL mutations. Nilotinib was demonstrated in a phase I study to be active against a small subset of imatinib-resistant Ph+ ALL adult pts. Based on this preliminary data, a phase II, open-label study was designed to further evaluate the safety and efficacy of nilotinib in pts with relapsed/refractory Ph+ ALL.Methods: The initial dose of nilotinib was 400 mg twice daily (BID) with escalation to 600 mg BID, when there was an inadequate response and no safety concerns.Results: The safety and efficacy data were reported for 41 Ph+ ALL pts in which 38 had relapsed and 3 were refractory [(37 had active disease; 4 had minimal residual disease (MRD)]. The median age at study entry was 46 (18–75) years and 54% were men. At study entry 31% of pts had >35% Ph+ metaphases; chromosomal abnormalities other than Ph+ were noted in 32% (13/41) pts, and extramedullary involvement was present in 3 pts. The median time since initial diagnosis of Ph+ ALL was 15 months. Treatment with nilotinib is ongoing for 2 pts (5%). Most discontinuations were due to disease progression (68%). The median treatment duration was 53 (1–563) days and the median dose intensity was 800 mg/day. Thirty-one percent of pts had >35% of Ph+ metaphases, chromosomal abnormalities other than Ph+ were noted in 32% of the pts, and extramedullary involvement was present in 7% of pts. Complete response (CR) was reported in 10 (24%) pts including 1 patient with MRD. Hematological improvement was observed in 1 (3%) pt, stable disease was observed in 9 pts, 14 pts had progressive disease. The most common Grade 3/4 laboratory abnormalities were thrombocytopenia (60%), neutropenia (43%), anemia (21%), and hypophosphatemia (22%).Conclusions: Nilotinib as a monotherapy appears to have promising activity and a favorable safety profile in relapsed/refractory Ph+ ALL pts. The use of nilotinib in combination with chemotherapy for Ph+ ALL merits further investigation.

Treatment Outcome with Imatinib-Based Maintenance Therapy in Elderly Patients with Philadelphia-Positive Acute Lymphoblastic Leukaemia (Ph+ALL) Ineligible for Allogeneic Stem Cell Transplantation (SCT).

Background: The tyrosine kinase inhibitor imatinib (IM), alone or in combination with induction and post-remission chemotherapy, has become the mainstay of front-line treatment for Ph+ ALL, followed by allogeneic SCT as a potentially curative treatment option. However, alloSCT is not feasible in many elderly or comorbid patients. In a prospective, randomized clinical trial of elderly patients with de novo Ph+ ALL, we recently demonstrated that IM induction followed by IM in combination with intensive consolidation chemotherapy of approximately one year duration was feasible in the majority of pts. but associated with a high relapse rate. To assess the impact of IM-based maintenance, we here provide an analysis of maintenance therapy with IM either alone, in combination with low dose interferon-α (LD-IFNα), or with zoledronic acid. In addition, we examined whether determination of minimal residual disease (MRD) and/or BCR-ABL mutation status prior to and during pre-maintenance therapy were predictive of freedom from relapse and remission duration.Patients and Methods: Remission induction and consolidation therapy have been reported previously (Ottmann et al., Cancer 109:2068–76, 2007). As the present analysis focuses entirely on the maintenance phase, patients who failed to achieve a CR, relapsed or died during the consolidation cycles are not included. Following a median of 6 cycles of remission induction and consolidation chemotherapy given concurrently with IM, 33 CR pts. (n=33; median age 69.5 yrs; [58–75 yrs.]) were enrolled either in a clinical trial of IM in combination with low-dose IFN (n=19), or with zoledronic acid (n=4), or received Glivec as a single agent (n=8). Minimal residual disease was serially assessed by quantitative RT-PCR and mutational analyses was performed by D-HPLC and direct sequencing.Results: With a median duration of maintenance of 17 months (range 6–72 mos.), 14 of 33 patients (42%) are in ongoing CR, with a median maintenance duration of 46 mos. (20–72 mos.). Median overall survival of all patients is 28 mos. (range: 9–81 mos.). Detection of a BCR-ABL mutation at or within 4 weeks of initial diagnosis was associated with inferior remission duration, 18 mos. versus 27 mos. (p=0.06). Remission was independent of the MRD response during induction and consolidation: 24 mos. and 26 mos. in pts. who did or did not achieve MRD negativity at any time; similarly, detection of MRD at the start of maintenance had no impact on time to progression.Conclusions: Among elderly Ph+ALL pts. who did not undergo SCT, treatment outcome with IM-based maintenance therapy is encouraging, but remissions are not sustained in the majority of patients. Surprisingly, persistent MRD positivity during consolidation or at the start of maintenance were not associated with an inferior outcome; conversely, relapses occurred even in patients who had achieved prolonged MRD negativity.

Bosutinib (SKI-606) Demonstrates Clinical Activity and Is Well Tolerated among Patients with AP and BP CML and Ph+ ALL.

Bosutinib (SKI-606) is an orally bioavailable dual Src/Abl inhibitor. Biochemical assays have shown it to be up to 200-fold more potent than imatinib as an inhibitor of Bcr-Abl phosphorylation. Unlike imatinib, bosutinib does not exhibit significant inhibition of c-kit or platelet-derived growth factor receptor (PDGFR), which may result in a relatively favorable safety profile. This is an ongoing open-label study in patients (pts) with Philadelphia chromosome positive (Ph+) accelerated phase (AP) and blast phase chronic myelogenous leukemia and (Ph+) ALL who failed prior imatinib therapy or other TKIs. Objectives are to assess safety and clinical activity of bosutinib. Pts receive bosutinib 500 mg/day. We report preliminary data for 57 pts, median age 54 yrs (range 22–83 yrs), 54% male. 23 pts (40%) were in AP, 15 (26%) in blast crisis (BC), 14 (25%) had Ph+ALL, and 5 (9%) were unclassified. Prior therapy included interferon (22 pts), imatinib (55 pts; data missing for 2 pts), dasatinib (17 pts), nilotinib (10 pts), stem cell transplant (5 pts). Overall median duration of bosutinib treatment was 2.7 mos (range 0.03–10.8 mos). Complete hematological response (CHR) was obtained in 7/25 evaluable pts (28%), including 4/14 (29%) pts with AP-CML, 2/8 (25%) pts with BC-CML, and 1/3 (33%) pts with Ph+ ALL. Among pts with no other TKI exposure, major cytogenetic responses (MCyR) were observed in 5/14 evaluable pts (36%), including 3/6 (50%) pts with AP-CML, 2/5 (40%) pts with BC-CML. Among pts with prior TKI exposure, 3/10 (30%) had MCyR, including 0/3 AP, 1/4 BP, and 2/3 ALL pts. Median time to MCyR was 8.9 weeks for pts previously exposed and 12 wks for unexposed to other TKIs. Duration of MCyR was 18 wks. 19 previously unexposed patients were evaluable for major molecular response. 4 (21%) had major molecular response, 3 (16%) of which were complete. Of 42 pts with samples tested for mutations, 13 different mutations were found in 20 pts (48%), including 5 cases of T315I. CHR occurred in 2/3 pts with P-loop mutations and 5/17 with non-P-loop mutations; MCyR occurred in 2/2 pts and 4/9 pts, respectively. Treatment was generally well tolerated in this cohort of heavily pretreated patients. The most common adverse events were gastrointestinal (diarrhea [56%], nausea [37%], vomiting [35%]) but these were usually grade 1–2, manageable and transient, reducing in frequency and severity after the first 3–4 weeks of therapy. Grade 3–4 hematologic laboratory abnormalities reported included thrombocytopenia in 31 pts (59%), neutropenia in 20 pts (38%), and anemia in 13 pt (25%). Grade 3–4 non-hematologic toxicities were diarrhea (9%) and vomiting (9%). Fluid retention was reported in 8 pts (14%), including 2 cases (3%) of pleural effusion (grade 2 and 3). Both were considered unrelated to treatment. Bosutinib is effective in imatinib-resistant pts with advanced CML. Responses were observed across a wide range of Bcr/Abl mutations. Bosutinib has a favorable toxicity profile with a small number of pts experiencing hematologic toxicity and fluid retention, possibly due to the lack of c-kit inhibition and PDGFR inhibition, respectively.

Dasatinib (SPRYCEL®) in Children and Adolescents with Relapsed or Refractory Leukemia: Preliminary Results of the CA180018 Phase I/II Study from the ITCC Consortium.

Outcome for children with relapsed/refractory leukemia is unsatisfactory. Dasatinib is a novel oral kinase inhibitor of BCR-ABL, KIT, and SRC family kinases. Recently, approval was granted for adult CML and Ph+ ALL with resistance to prior therapy. CA180018, the 1st trial evaluating dasatinib in children (age 1–20 yrs) and conducted in 12 centres from the ITCC Consortium, is a phase I/II dose-finding study in patients (pts) with either imatinib resistant/intolerant CML, relapsed/refractory Ph+ or Ph− ALL, or AML in 2nd or greater relapse. The starting dose was 60 mg/m2 QD; intra-pt dose escalation for lack of initial response and dose-(de)escalations were safety and efficacy guided and aimed at establishing a safe and effective dose for each leukemia sub-type. These data reflect the first 34 pts (median age 12.6 y, range 1.6–20) treated from Mar 06 to Aug 07: 6 chronic phase [CP] CML, 2 advanced phase [AP] CML, 8 Ph+ ALL, 6 Ph− ALL, and 12 Ph− AML. Prior therapy included chemotherapy, imatinib, and stem cell transplant. Intra-pt dose escalation to 80 or 100 mg/m2 occurred in 21 pts. 7 pts remain on study. Safety: 51 SAEs in 22 patients were noted, and included febrile neutropenia/infections (n=27), GI toxicity (n=8), rash (n=4), and grade 3/4 thrombocytopenia (n=3). One dose-limiting toxicity (DLT) was observed: a grade 4 anaphylactic reaction 5 h after the first dose in a pt with a history of hypensensitivity reactions. One pt had a pleural effusion that also contained leukemic cells. Responses: 5/6 CP CML pts are evaluable for response: 3 pts went into complete hematologic remission; 2 had a complete and 1 had a partial cytogenetic response (CygR). One pt was enrolled with molecular disease only, and PCR values became undetectable after 3 months. One pt never achieved a hematological or CygR, yet no resistance mutations were detected. The pt with myeloid AP CML had a major CygR for ∼3 months; the pt with lymphoid BP CML had a complete CygR (14% BCR-ABL+ cells using FISH), but experienced clonal evolution with ∼28% blasts in the marrow at the same time. Of the 8 pts with Ph+ ALL, 3 showed complete morphologic and cytogenetic remission, lasting for 6 weeks, 1 month, and >2 months for one pt still on study. One other pt with isolated CSF relapse cleared the CSF for ∼7 months with dasatinib only, has subsequently received additional intrathecal chemotherapy, and remains on study 17 months later. Four pts did not respond, 2 of who harbored the T315I mutation. Of the 6 Ph− ALL pts, 4 were evaluable, and none achieved a CR. In 1/4 pts there was a significant drop in WBC for approximately 2 months. Among the 12 Ph− AML pts, one had a drop in BM blasts from 60 to 2% (without full PB regeneration), and one pt from 60 to 15% blasts. None of the AMLs appeared to be KIT-mutated. These data show that dasatinib was well tolerated up to 100 mg/m2; the encouraging early signs of response in Ph+ disease support its further exploration in relapsed/refractory pediatric leukemia pts. Higher dose levels are currently being explored and updated clinical and PK data will be presented.

A phase II study of nilotinib administered to imatinib resistant or intolerant patients with chronic myelogenous leukemia (CML) in blast crisis (BC) or relapsed/refractory Ph+ acute lymphoblastic leukemia (ALL)

7040 Background: Nilotinib is a highly selective Bcr-Abl tyrosine kinase inhibitor that is 30-fold more potent than imatinib. In a phase I trial, nilotinib demonstrated efficacy and favorable tolerability in these pts. These results expand upon the phase I experience Methods: This phase II open-label study was designed to evaluate the safety and efficacy of nilotinib in adult imatinib-resistant or - intolerant BC pts or pts with relapsed/refractory Ph+ALL. Primary endpoint was investigator assessment of best hematologic response for BC and complete response for Ph+ALL pts. Nilotinib was started at 400mg BID with escalation to 600mg BID if no adequate response. Results: Safety and efficacy data are reported for 120 BC (27 lymphoid, 87 myeloid, 6 unknown) and 41 Ph+ALL pts (37 active disease, 4 residual disease, 38 relapsed, 3 refractory). 60% of pts had &gt;35% Ph+ metaphases for BC and 31% for Ph+ALL. Median ages was 54 yrs for BC and 46 yrs for Ph+ALL pts. Chromosomal abnormalities other than Ph+ were noted in 64 (53%) BC and 12 (29%) Ph+ALL pts. Extramedullary involvement was present in 44 (37%) BC and 3 (7%) Ph+ALL pts. Treatment is ongoing for 21 (18%) BC and 4 (10%) Ph+ALL pts. Majority of discontinuations were due to disease progression [61 (51%) in BC; 26 (63%) in Ph+ALL). Median treatment duration was 53 (1–441) and 72 (3–363) days for BC and Ph+ALL, respectively. Median dose intensity was 800mg/day for both pt groups. CHR was reported in 25 (21%) pts, marrow responses in 7 (6%) pts, and return to chronic phase in 10 (8%) pts. Complete response was reported in 10 (24%) Ph+ALL; of which, 1 patient had minimal residual disease. The most common Grade 3/4 AEs were thrombocytopenia (41%), neutropenia (28%), pneumonia (11%), and anemia (27%) in BC and thrombocytopenia (24%) in Ph+ALL pts. During study period death occurred in 9 (8%) BC and 3 (7%) Ph+ALL pts. No Ph+ALL pt developed CNS disease while on therapy. Conclusions: Nilotinib has significant clinical activity and is well tolerated in imatinib-resistant or -intolerant BC and relapsed/refractory Ph+ALL pts. Nilotinib represents an important new treatment option for these pts in which there remains a high unmet medical need. No significant financial relationships to disclose.