Chronic Myeloid Leukemia In Lymphoid Blast Crisis Research Articles

A Phase I Study of Asciminib (ABL001) in Combination with Dasatinib and Prednisone for BCR-ABL1-Positive ALL and Blast Phase CML in Adults

Introduction:Oral ABL1 kinase inhibitors rapidly produce deep remissions in BCR::ABL1+ acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphoid blast crisis (CML-LBC). Second generation TKIs such as dasatinib (DAS) are more effective than imatinib (Foa et al. Blood 2011) but patients may develop resistance. Asciminib (ASC), previously ABL001, is a STAMP (Specifically Targeting the ABL Myristoyl Pocket) allosteric ABL1 inhibitor that binds to a site spatially distinct from ATP-competitive TKIs. Combination treatment with an allosteric and an ATP-competitive TKI may deepen clinical responses and limit mutational resistance as supported by a cell line xenograft model of CML (Wylie et al. Nature 2017) and patient-derived xenograft models of BCR::ABL1+ ALL. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and active in BCR::ABL1+ ALL and CML-LBC. Methods: This investigator initiated, phase I study (NCT03595917) of asciminib (ASC) in combination with DAS plus prednisone (pred) for BCR::ABL1+ ALL studied ASC in escalating doses in a 3+3 design with the primary objective to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D); a 10 patient (pt) expansion cohort was then accrued. Secondary objectives define the depth and durability of responses. Pts with BCR::ABL1+ ALL or CML-LBC newly diagnosed ≥ 50 years (yrs), or unfit; or relapsed/refractory (no prior DAS or ASC) were eligible in dose escalation; all pts ≥18 yrs were eligible in dose expansion. Pts received DAS 140 mg/day(d) and pred 60 mg/m 2/d 1-24 (max 120 mg/d, tapered d 25-32) with escalating daily doses of ASC (DL1: 40 mg; DL2: 80 mg; DL3: 160 mg). DAS and ASC were given in 28-d cycles indefinitely in setting of clinical benefit, with allogeneic stem cell transplant (SCT) consolidation after d85 per treating physician. Dose-limiting toxicity (DLT) was initially defined as CTCAEv4 non-heme toxicity grade (gr) 3+; the study was amended to assess DLT via CTCAEv5. Correlative science efforts seek to define biomarkers of response and resistance to dual ABL1 blockade. Results:The study enrolled 25 pts (48% female), with 14 in dose escalation (13 evaluable), and 11 in expansion (10 evaluable). Most (92%) had new ALL (p190: 15/23, p210: 8/23; 8% had CML-LBC. Median age was 65 yrs (range 33 - 85; 8 pts 70+). Median WBC was 15.1 K/μL (range 0.9 - 251.9 K/μL). No pt had CNS disease. DL1 and DL2 enrolled 3 pts each without DLT. Two of 3 pts at DL3 developed asymptomatic CTCAEv4 gr 3 amylase elevation during C1 meeting original DLT criteria. A 4 th pt enrolled on re-opened DL3 under an amendment defining asymptomatic CTCAEv4 gr 3 amylase/lipase elevations persisting ≤5d to not be a DLT. This pt experienced an asymptomatic CTCAEv4 gr 3 lipase elevation meeting DLT criteria. Thus, the study de-escalated to DL2, re-opening under an amendment assessing DLTs via CTCAEv5. Of note, all DLTs at DL3 would be gr <3 per CTCAEv5. Four more pts (3 evaluable) enrolled at DL2 without a DLT. Thus, ASC 80 mg/d was declared the RP2D, and 11 pts age ≥18 yrs enrolled in an expansion cohort, 10 of whom initiated study treatment. No pt had symptomatic pancreatitis. Rates of molecular response after 3 cycles among all evaluable patients were 58.3% (14/24) for MRD 3.0 and 25.0% (6/24) for MRD 4.0. Evaluable pts with new ALL treated at the RP2D achieved molecular response rates after 3 cycles of 78.6% (11/14) for MRD 3.0 and 42.9% (6/14) for MRD 4.0. Both patients with imatinib-refractory CML-LBC progressed (C9, C3). Of those with ALL, 8 bridged to SCT after 2-8 cycles; 2 elected local care (C5, C7); 1 transitioned to ponatinib for inadequate response plus recurrent DAS pulmonary toxicity (C4); 6 remained on study treatment until disease progression (C4 - MRD+, C45, C11, C11); 3 remain on study. Correlative science evaluation of serial pt biospecimens in pursuit of predictive biomarkers will be shared at the meeting. Conclusion: Dual ABL1 kinase inhibition with ASC and DAS plus pred in BCR::ABL1+ ALL and CML-LBC is feasible and tolerable in adults with BCR::ABL1+ ALL and CML-LBC. DLTs at ASC 160 mg/d were asymptomatic amylase and lipase elevation, without clinical sequelae. ASC 80 mg/day was declared the RP2D, and an expansion cohort of 10 pts was completed. High rates of molecular response and bridging to transplant highlight encouraging preliminary activity. A phase II expansion cohort incorporating blinatumomab is now open.

BCR::ABL1-Positive Acute Lymphoblastic Leukemia: Different Clinical Behavior and Relevance of Prognostic Features in Typical ALL and in CML-like Disease

Recently we showed that in 20-30% of children diagnosed as BCR::ABL1-positive (Ph+) acute lymphoblastic leukemia (ALL) the BCR::ABL1 fusion is present in a wider clone, involving myeloid cells, non-ALL B-cells and T-cells. This leads to discordant minimal residual disease (MRD) levels assessed by the quantification of two available MRD targets - immunoglobulin (IG)/T-cell receptor (TR) genes rearrangements and BCR::ABL1 fusion. As the cases with multilineage BCR::ABL1 involvement resemble lymphoid blast crisis (LBC) of chronic myeloid leukemia (CML) we named these leukemias "CML-like", as distinct from "typical Ph+ ALL". To reveal biological and clinical differences between the two subtypes, we retrospectively analyzed prognostic relevance of MRD and other features in 147 children with BCR::ABL1-positive ALL treated according to EsPhALL (n = 133) or other (n = 14) protocols. MRD was assessed by DNA-based monitoring of BCR::ABL1 genomic breakpoint and of clonal IG/TR rearrangements. Although overall prognosis of CML-like (n = 48) and typical Ph+ ALL (n = 99) was similar (5-year EFS 60% and 49%; 5-year OS 75% and 73%, respectively), typical Ph+ ALL presented more relapses (12/48 vs. 42/99; p = 0.046) while CML-like patients more often died in the first remission (6/48 vs. 3/99; p = 0.059 for CR1 deaths in total and p = 0.01 for CR1 deaths out of the total deaths). Prognostic role of MRD measured at standard treatment timepoints (TP1 - end of induction IA, day 33; TP2 - end of consolidation IB, week 12) was highly significant in the typical Ph+ ALL (p = 0.0005 for EFS). In contrast, in CML-like patients prognostic impact of MRD was not significant and thus inapplicable for therapy adjustment. Similarly, although diagnostic white blood cell counts were similar in the two subgroups, impact of the hyperleukocytosis ≥ 50 x 109/l on outcome was highly significant in the typical Ph+ ALL (and remained significant in multivariate analyses, being apparent even in patients with fast MRD response), but no prognostic impact was found in CML-like patients. While in the typical Ph+ ALL the NCI risk tended to impact 5-year OS (89% vs. 67%; p = 0.03), there was no impact on prognosis in the CML-like patients. The BCR::ABL1 transcript type (minor/p190 vs. Major/p210), IKZF1 deletion, sex, or the age at diagnosis (< 10 vs. ≥ 10 years) did not show any prognostic relevance. Earlier start of continuous tyrosine kinase inhibitors (TKI) treatment (day 15 vs. day 33) was associated with lower MRD levels in the typical Ph+ ALL but not in CML-like patients. However, earlier TKI start tended to result in better 5-year OS in both subtypes (78% vs. 67%; p = 0.082 and 83% vs. 60%; p = 0.066 for typical Ph+ ALL and CML-like, respectively). To shed more light on the nature of the two subtypes, we now collect additional data (including transcriptome profiles, single cell characteristics, genomic breakpoint features) in order to reveal biological background, cell of origin and further potential differences between the subtypes. Such data could help to find optimal treatment strategies, and also to answer the attractive question whether CML-like disease is in fact a regular CML in LBC or whether there is a wider spectrum of BCR::ABL1-positive leukemias. If these studies lead to a conclusion that CML-like is indeed indistinguishable from LBC-CML, it will be necessary to re-define CML as a disease manifesting - at least in childhood - very often in LBC and with the minor-BCR::ABL1/p190 fusion variant. In conclusion, our data unequivocally confirm that although clinically presenting at diagnosis as one entity, there are two biologically distinct subtypes of BCR::ABL1-positive ALL - typical Ph+ ALL and CML-like disease. The different biology is reflected in treatment response and although the overall survival rates of both subtypes are similar on current protocol, the key reasons of treatment failure are different - toxicity of intensive ALL treatment in CML-like patients and relapses in typical Ph+ ALL. Thus, early distinguishing of the two subtypes is essential to enable optimal treatment approach and therapy adjustments in upcoming trials. For the typical Ph+ ALL, TKI and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, representing one-fourth to one-third of childhood patients diagnosed as BCR::ABL1-positive ALL, no relevant risk factor applicable for therapy tailoring was found so far.

Safety and Efficacy of Blinatumomab in Combination with a Tyrosine Kinase Inhibitor for the Treatment of Relapsed Philadelphia Chromosome-Positive Leukemia

Safety and Efficacy of Blinatumomab in Combination with a Tyrosine Kinase Inhibitor for the Treatment of Relapsed Philadelphia Chromosome-Positive Leukemia

Outcome of Patients with Chronic Myeloid Leukemia in Lymphoid Blast Crisis (CML-LBC) and Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) Treated with Hyper-CVAD and Dasatinib

Outcome of Patients with Chronic Myeloid Leukemia in Lymphoid Blast Crisis (CML-LBC) and Philadelphia Chromosome (Ph)-Positive Acute Lymphoblastic Leukemia (ALL) Treated with Hyper-CVAD and Dasatinib

Blinatumomab in Combination with Tyrosine Kinase Inhibitors Safely and Effectively Induces Rapid, Deep, and Durable Molecular Responses in Relapsed and Refractory Philadelphia Positive Acute Leukemias

Background The addition of tyrosine kinase inhibitors (TKIs) to chemotherapy improves the prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Single agent blinatumomab, a bi-specific T-cell engaging CD3-CD19 antibody construct, is more effective than multi-agent chemotherapy in relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and successfully achieves minimal residual disease (MRD) negativity in MRD positive patients. Limited data is available on the safety and efficacy of these agents in combination and uncertainties remain regarding the optimal use of blinatumomab +TKIs. Dual therapy with blinatumomab and TKIs may offer a safe and effective treatment option for relapsed and refractory patients without the incorporation of chemotherapy. Patients and Methods We report our retrospective experience in 18 patients who received blinatumomab +TKI for relapsed/refractory (R/R) Ph+ ALL (n=14), MRD+ Ph+ ALL (n=2) and chronic myeloid leukemia in lymphoid blast crisis (CML-LBC) (n=2). Ten patients received ponatinib; 8 patients received second generation TKIs (dasatinib, n=5; nilotinib, n=2; bosutinib, n=1). Blinatumomab was administered according to the package insert and TKI was initiated on day 1. Median follow up was 15 months and median number of blinatumomab +TKI cycles was 2 (range, 1-5). The median age was 47 years (range, 22-77). The median number of prior therapies was 2 (range, 1-8) and the median number of prior TKIs was 2 (range 1-4). Preexisting T315I mutations were present in one third of patients and 5 patients had relapsed following allogeneic stem cell transplant (alloHSCT). Results Transient grade 2-3 transaminase elevation was observed in 3 patients (ponatinib=2, dasatinib=1). Cytokine Release syndrome was observed in 2 patients. Transient neurotoxicity was observed in 3 patients (1patient with grade 3). All grade 2 and 3 toxicities were observed during the first cycle and all patients were successfully able to complete induction with blinatumomab +TKI. One patient on dasatinib experienced a PICC-associated DVT. There were no other vascular events observed. For patients with r/r disease the CR rate was 88% (14/16) with most of these remissions being MRD negative by flow and PCR (12/14). Both of the patients treated for MRD positivity achieved an MRD negative response. Among T315I mutated patients, 5/6 achieved a CR and 4/5 were MRD negative. The median time to remission was 35 days. The 1 year survival was 80% and the median overall survival following blinatumomab therapy was 45 months. Among 16 pts who achieved an initial response to blinatumomab +TKI, six have expired at the time of this analysis. At the time of relapse on combination therapy, 3/8 pts exhibited new T315I mutations. Of note, all of these were treated with blinatumomab in combination with a second generation TKI. All patients maintained their CD19 positivity at the time of relapse. Both pts with CML-LBC were able to move to a transplant following therapy with blinatumomab +TKI and both attained MRD negative status as measured by Bcr/Abl PCR values. In total, 10 patients treated with blinatumomab +TKI proceeded to alloHSCT, 7 of these are still alive. Median duration of response (DOR) was 5.1 months with maximum DOR of 23.3 months. Discussion While limited due to its retrospective nature and relatively small sample size, this data set begins to answer important regarding the safety and efficacy of blinatumomab +TKI. Combination therapy was effective at achieving rapid and deep responses in this refractory patient population. The toxicities observed with blinatumomab +TKI were in keeping with those seen with blinatumomab alone. However, care should be taken in regard to potential overlapping toxicities of TKIs and blinatumomab, such as hepatotoxicity. The study also suggests a role for blinatumomab treatment in patients with CML-LBC, a rare and difficult disease state to treat. The identification of T315I mutation at relapse in 40% of patients on blinatumomab +second generation TKIs supports further prospective studies with ponatinib and blinatumomab in combination. Finally, loss of C19, which has relevance with regards to the potential benefit of subsequent CD19-directed therapies such as chimeric antigen receptor T-cell therapy was not seen in patients treated with blinatumomab +TKI. Disclosures McCloskey: Jazz: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau; Novartis: Speakers Bureau. Gagnon:Amgen: Speakers Bureau. Goldberg:Cancer Outcomes Tracking and Analysis (COTA) Inc.: Equity Ownership; Bristol-Myers Squibb: Consultancy; COTA: Equity Ownership. Koprivnikar:Amgen: Speakers Bureau; Pfizer: Honoraria; Novartis: Speakers Bureau; Abbvie: Speakers Bureau.

A Novel Approach to Identify a Putative Leukemia Stem Cell Population in Acute Lymphocytic Leukemia (ALL) and Distinguish Philadelphia Chromosome-Positive ALL from Lymphoid Blast Crisis Chronic Myeloid Leukemia

Introduction: Recent evidence supports the clinical significance of leukemia stem cells (LSCs) in acute myeloid leukemia (AML). However, the identification of LSCs in acute lymphocytic leukemia (ALL) has proved challenging, as transplantation studies in immunocompromised mice have yielded conflicting results. The distinction between Philadelphia chromosome-positive (Ph+) ALL and lymphoid blast crisis (LBC) chronic myeloid leukemia (CML) is also controversial. We previously identified a clinically relevant CD34+CD38- population of LSCs with intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity (CD34+CD38-ALDHint) in AML [Gerber, et al. Blood, 2012]. This population was not present in healthy controls and could be distinguished from normal hematopoietic stem cells (HSCs), which had higher levels of ALDH activity (CD34+CD38-ALDHhigh). We hypothesized that the same approach could be used to identify a putative LSC population in ALL. Furthermore, in contrast to most cases of AML, the chronic phase CML stem cell was found to reside in the same CD34+CD38-ALDHhigh population as normal HSCs [Gerber, et al. Am J Hematol, 2011]. We therefore also hypothesized that the presence of BCR/ABL mutations in the CD34+CD38-ALDHhigh population might help distinguish LBC CML from Ph+ ALL.Methods: Bone marrow and/or peripheral blood specimens were collected at diagnosis from patients with B cell ALL or LBC CML on an IRB-approved protocol. A total of 7 patients were evaluated: 2 Ph- ALL, 2 Ph+ ALL, and 3 LBC CML patients. CD34+ cells were isolated by magnetic bead and column selection, then analyzed by flow cytometry with respect to CD38 expression and ALDH activity. Sorted cell populations were analyzed by fluorescence in situ hybridization (FISH) for leukemia-specific abnormalities. Polymerase chain reaction was performed on clinical samples to determine the presence of a p190 vs. p210 transcript.Results: All patients harbored an aberrant CD34+CD38-ALDHint population, similar to that previously seen in AML. This population was ≥95% positive for BCR/ABL by FISH in all Ph+ ALL and LBC CML cases. It was similarly positive (≥75%) for other leukemia-specific FISH abnormalities (including trisomy 4, 8, 10, 12, and/or 21) in all four ALL cases, as well as one LBC CML case. Conversely, the CD34+CD38-ALDHhigh population (which typically contains the normal HSCs) lacked any of the other cytogenetic abnormalities in all of the cases, irrespective of Ph status or a diagnosis of ALL vs. CML. Notably, the CD34+CD38-ALDHhigh population was negative for BCR/ABL in the Ph+ ALL cases but was >95% positive for BCR/ABL by FISH in the LBC CML cases. The B cell differentiation marker, CD19, was expressed on the CD34+CD38-ALDHint but not the CD34+CD38-ALDHhigh population in all ALL cases, both Ph- and Ph+. In contrast, CD19 expression was variable in the LBC CML cases. Both Ph+ ALL cases possessed a p190 BCR/ABL transcript, whereas all of the LBC CML cases contained a p210 transcript. Also of note, the CD34+CD38-ALDHint population was persistently detectable in one of the LBC CML patients while in complete remission after induction therapy; that patient subsequently relapsed.Conclusions: An abnormal CD34+CD38-ALDHint population was identified in all cases of B cell ALL and LBC CML. This population is analogous to a previously identified, clinically relevant LSC population in AML and may represent a putative LSC population in ALL. The CD34+CD38-ALDHhigh population was normal by FISH in the ALL cases but contained the BCR/ABL mutation in the LBC CML cases, thus permitting distinction between Ph+ ALL and LBC CML (which also differed based on the presence of p190 vs. p210 transcripts, respectively). Additionally, clonal evolution from chronic phase to lymphoid blast crisis CML was apparent, based on the acquisition of additional cytogenetic abnormalities unique to the CD34+CD38-ALDHint population as compared to the CD34+CD38-ALDHhigh population. The presence of CD19 on the putative LSCs in the four cases of ALL suggest that CD19-directed therapies may target the LSCs and thus may have curative potential in those cases. This assay may serve as a means to evaluate other possible therapeutic targets. Lastly, the detection of the abnormal CD34+CD38-ALDHint population may have utility as a minimal residual disease assay for monitoring response to treatment. These findings warrant validation in a larger patient cohort. DisclosuresGerber:Janssen: Research Funding; Alexion: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Grunwald:Alexion: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medtronic: Equity Ownership; Janssen: Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding. Avalos:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Pediatric chronic myeloid leukemia with B-cell lymphoid blast crisis at presentation

TO THE EDITOR: Chronic Myeloid Leukemia (CML) is a rare hematological malignancy accounting for less than 3% of pediatric and adolescent leukemias, with an annual incidence of approximately 1 per million children and young people aged 20% blasts in the marrow or the presence of extra-medullary blast proliferation [3, 4]. Blast transformation of CML is lymphoid in 30% of cases and myeloid in the remaining 70%. We report the case of a 10-year-old boy who presented with a 6-week history of weight loss and left-sided upper abdominal pain. On examination, he was found to have substantial splenomegaly extending into the right iliac fossa. His full blood count showed a hemoglobin level of 6.6 g/dL, a platelet count of 148×109/L, and a total white cell count of 575×109/L. A differential count showed the following: blasts, 30%; promyelocytes, 6%; myelocytes, 24%; metamyelocytes, 11%; neutrophils, 7%; eosinophils, 9%; basophils, 3%; monocytes, 1%; and lymphocytes, 10% (Fig. 1). Flow cytometry of the blast population showed that the blasts expressed CD19, cyCD79a, CD10, HLA-DR, CD34, and TdT surface antigens. Chromosome analysis showed a t(9;22)(q34;q11) translocation consistent with a Philadelphia chromosome (Fig. 2A). Interphase fluorescence in situ hybridization (FISH) showed BCR-ABL1 fusion signals in 90% of the nuclei (Fig. 2B). A minor breakpoint cluster region (210 kDa) was confirmed using RT-PCR. In the absence of a documented CML-CP, distinguishing between lymphoid blast crisis of CML and a Philadelphia chromosome-positive ALL can be difficult. A diagnosis of CML in B-cell lymphoid blast crisis rather than de novo precursor B-cell ALL was made because of the concurrent presence of basophilia, a predominance of metamyelocytes and myelocytes, and the p210 BCR-ABL transcript. The patient did not have any additional chromosomal anomalies associated with advanced phase CML and was negative for IgH rearrangement on FISH analysis. Fig. 1 Blood film demonstrating chronic myeloid leukemia in lymphoid blast transformation. Fig. 2 (A) Karyotype showing t(9;22)(q34;q11) Philadelphia translocation. (B) Interphase FISH, Vysis dual-fusion probe set. Green: BCR; red: ABL1; yellow: fused BCR and ABL signals corresponding to der(9) and der(22) translocation products. The patient achieved a complete hematological response and a minor cytogenetic response following induction therapy comprising dexamethasone, vincristine, daunorubicin, asparaginase, and imatinib. Nonetheless, he subsequently died of idiopathic pneumonitis after allogeneic stem-cell transplantation. The progression from CML-CP to CML-BC in the pre-tyrosine kinase era is well described, but CML-BC at presentation in children is extremely rare. The case reported here adds to the literature on the simultaneous presence of features of both lymphoblastic transformation and CML at presentation in the absence of cytogenetic clonal evolution. Further research into the biology of the aggressive phase of CML is required to develop novel targets to improve outcomes.

Phase 1 Dose-Escalation Study of PF-04449913, An Oral Hedgehog (Hh) Inhibitor, in Patients with Select Hematologic Malignancies

Abstract 424 Background:Hh signaling is activated in a variety of human cancers and is required for maintenance of leukemic stem cell (LSC) populations in several hematologic malignancies. PF-04449913 selectively inhibits Hh signaling by binding Smoothened, a membrane protein that regulates the Hh pathway. PF-04449913 significantly reduced LSC burden following xenotransplantation of patient-derived CD34+ imatinib-resistant chronic myeloid leukemia (CML) cells in preclinical models. Methods:This first-in-patient Phase 1a dose-escalation study is assessing first-cycle dose-limiting toxicities (DLTs) and the recommended Phase 2 dose (RP2D) of PF-04449913 in patients with select hematologic malignancies (primary endpoint). Secondary endpoints include safety, pharmacokinetics (PK), pharmacodynamics, and preliminary signs of efficacy as defined by disease-specific guidelines. Patients had refractory, resistant, or intolerant select hematologic malignancies and could be previously untreated but not candidates for standard therapies: CML including T315I mutations (any phase), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelofibrosis (MF), or chronic myelomonocytic leukemia (CMML). Cohorts of ≥3 patients received PF-04449913 alone, administered continuously in 28-day cycles, starting at a dose of 5 mg orally once daily. Results:Thirty-two patients have been enrolled at doses up to 270 mg: 18 males/14 females; AML, 18; MF, 6; CML, 5; MDS, 3; with a median age of 68.5 (35–79) years. ECOG PS was 0/1/2: n=11/16/5. Treatment duration ranged from 1 to 387 days (AML: 1–266; CML: 1–281; MDS: 2–335; MF: 44–387 days). One patient discontinued the study due to a treatment-related adverse event (AE) after 137 days of therapy at the 10 mg dose level (grade [G] 3 hemorrhagic gastritis in the setting of chronic proton pump inhibitor administration prior to and during the study). One AML patient evolved from CMML on 80 mg had a DLT comprising G3 hypoxia and G3 pleural effusion. The majority of AEs were of G1/2 severity; the most frequent treatment-related AEs included dysguesia (16%), alopecia (6%), arthralgia (6%), decreased appetite (6%), nausea (6%), and vomiting (6%). Preliminary indications of efficacy were observed across all hematologic diseases studied. One patient with AML (evolved from CMML and with a concurrent diagnosis of systemic mastocytosis) achieved a complete remission with incomplete blood count recovery; bone marrow blast count decreased from 92% to 1%. Five AML patients had a ≥50% reduction in bone marrow blast counts (20% to 10%, 70% to 20%, 44% to 8%, 14% to 7%, 40% to 10%). One patient with low-risk MDS, currently remaining on study after 335 days, achieved significant reduction in spleen size and a hematologic improvement in platelets (from 98.5 to 369 ×109/L) and neutrophils (ANC from 410 to 5490), and is no longer granulocyte colony-stimulating factor (G-CSF) dependent. Five patients with MF attained stable disease; an additional MF patient, currently remaining on study after 385 days, achieved clinical improvement with a >50% reduction in extramedullary disease (spleen size decreased from 10 cm to 3.5 cm sustained over 8 weeks). One patient with T3151 lymphoid blast crisis CML on study for 115 days achieved a major cytogenetic response with loss of their T3151 mutation. PF-04449913 PK data (5–180 mg) indicated a dose-proportional profile, with a median time to maximum concentration (Tmax) of 1–2 hours, a mean half-life ranging from 17 to 35 hours, and a large volume of distribution (mean range 250–480 L). Following daily dosing, steady state was achieved by Day 8 and the median accumulation ratio ranged from 1.3 to 2.9. Conclusions:PF-04449913 was safe and well tolerated, with early signs of efficacy observed in all hematologic diseases studied. Several patients with aggressive malignancies remained on trial for prolonged durations with improved quality of life; some exhibited cellular differentiation as determined by flow cytometry. On-target AEs (e.g. dysgeusia and alopecia) were observed at multiple dose levels. Pharmacokinetics were linear, predictable, and compatible with once-daily dosing. On the basis of these encouraging results, a Phase 1b study of PF-04449913 in combination with bosutinib and dasatinib is planned in patients with CML. Disclosures:Jamieson:Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria; Wintherix: Equity Ownership. Cortes:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Pfizer Oncology: Research Funding; Novartis: Research Funding; BMS: Research Funding; Infinity: Research Funding. Oehler:Pfizer Oncology: Research Funding. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Zhang:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Shaik:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Martinelli:Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

A Pilot Study of the Combination of Nilotinib and Hyper-CVAD for Philadelphia Chromosome Positive Acute Lymphocytic Leukemia and Lymphoid Blast Crisis Chronic Myelogenous Leukemia

AbstractAbstract 2144 Background:Nilotinib is a second generation bcr-abl tyrosine kinase inhibitor (TKI) that has at least 30 times the potency of Imatinib (IM). Nilotinib has been approved by the FDA for the treatment of adults with chronic myeloid leukemia (CML) in chronic phase (CP) in the frontline, and for CP or accelerated phase (AP) who are either resistant or intolerant to prior therapies, including IM. Philadelphia chromosome positive (Ph+) acute lymphocytic leukemia (ALL) and lymphoid blast crisis of CML (Ly-BC) is aggressive and rapidly fatal. Complete remissions rates are low and have short durability. Relapse is inevitable unless patients (pts) receive an allogeneic stem cell transplant (SCT). The use of TKIs against Ph+ ALL or Ly-BC has shown limited durability of response as single agents. The transient nature of response to TKI is because only some of the blasts are still sensitive to bcr-abl inhibition, while the rest of the clones are not and require the addition of chemotherapy. Hyper-CVAD combined with the first generation TKI, IM is one such chemotherapy regimen that has been used in the management of adults with Ph+ ALL or Ly-BC (Thomas D, et al. Blood, 2004). Despite achieving >90% complete remission (CR) during induction phase, some pts have resistant disease or relapse shortly after attaining remission. We hypothesized that combining nilotinib with hyper-CVAD would achieve greater rates of sustained CR which ultimately translate into lower relapse rates, superior disease free survival, long term survival, and potential cure post SCT. The goal of our pilot case series was to study the feasibility of combining the more potent TKI, nilotinib to hyper-CVAD in pts with Ph+ ALL or Ly-BC, so as to improve the efficacy. Methods:Our case series involves 5 pts at our institution diagnosed with either Ph+ ALL or Ly-BC treated with a combination of hyper-CVAD and nilotinib given orally 400mg twice daily (initiated the day after completion of chemotherapy until the start of next cycle). After completion of eight cycles of chemotherapy, pts received either SCT or maintenance POMP in combination with nilotinib. Supportive care included prophylactic antibiotics, granulocyte colony stimulating factor, and transfusions as needed. None of them had central nervous system (CNS) involvement, but received intrathecal prophylaxis. Clinical characteristics at baseline, safety assessments, and efficacy evaluations including morphologic, cytogenetic, and molecular responses were analyzed. Results:All 5 pts (p190 Ph+ ALL-4, p210 Ly-BC-1; all males; Hispanic-2, Caucasian-3) received the combination for a median of 5 months. The median age was 29 (range 22–72) and the median follow-up period was 14 months. The Ly-BC pt was treated upfront with IM during CP and achieved major molecular remission (MMR) for 39 months, however due to noncompliance, relapsed with progression. All 5 patients achieved complete remission (CR), 60% after the first course. All of them achieved complete cytogenetic remission (CCyR) while 4 achieved MMR. Of the 2 Ph+ ALL pts who completed eight cycles, 1 received SCT and is in MMR for 20 months, while the other pt remains in MMR on POMP-nilotinib maintenance for over 19 months. Another pt is in MMR for 5 months. Two patients relapsed and died: the Ly-BC pt who achieved CR (without MMR) for 3 weeks and another pt that was in CCyR and MMR for 11 months. Most common adverse events included pancytopenia, fever, and infections (with no treatment related life threatening events). Conclusions:Our pilot study is the first to demonstrate the feasibility of combining nilotinib with hyper-CVAD. The results thus far are encouraging and will be expanded into a larger investigator initiated phase II study that can potentially prevent the emergence of resistant clones and improve survival in patients with Ph+ ALL or Ly-BC.Table 1ParameterHyper-CVAD + NilotinibNo. treated5Performance, ECOGn (%)04 (80%)1 to 21 (20%)WBC count at diagnosisn (%)30 or higher2 (40%)Platelet count at diagnosisn (%)Lower than 503 (60%)Responsen (%)CR5 (100%)Resistant0 (0%)Death2 (40%)Courses to CRn (%)13 (60%)21 (20%)More than 21 (20%)Adverse Events (Grade)n (%)Pancytopenia (4)5 (100%)FUO (2)2 (40%)Bacteremia (3)2 (40%)PNA (3)1 (20%)Cellulitis (2)1 (20%)Transaminitis (2)2 (40%) Disclosures:Off Label Use: Pilot study of Nilotinib and hyper-CVAD in Ph+ ALL.

A Comprehensive Deep-Sequencing Study of Blast Crisis Chronic Myeloid Leukemia (CML) Reveals New Insights Into Molecular Heterogeneity and Detects Mutations In 12 Different Genes In 82.5% of Cases

AbstractAbstract 884Blast crisis is the terminal phase of chronic myeloid leukemia (CML) with a short median survival of approximately six months. At present, little is known about molecular mechanisms underlying disease progression. We hypothesized that mutations occurring in other myeloid and lymphatic malignancies are acquired during disease progression from chronic phase to blast crisis. Here, in total 40 blast crisis CML cases (n=25 myeloid, n=10 lymphoid, n=5 not specified) were analyzed, all diagnosed between 9/2005 and 7/2009. First, all cases were investigated for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2–7, or exon 4–7 as published by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, in 17.5% (7/40) of cases intragenic IKZF1 deletions were detected. Secondly, next-generation deep-sequencing (454 Life Sciences, Branford, CT) was used to investigate 11 candidate genes in all 40 patients for a broad molecular screening. Known hotspot regions were sequenced for CBL (exons 8 and 9), NRAS (exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4), and NPM1 (exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1, and TP53. To perform this comprehensive study, amplicon-based deep-sequencing was applied using the small volume Titanium chemistry assay. To cope with the great number of amplicons, in total 59, 48.48 Access Arrays were applied (Fluidigm, South San Francisco, CA), amplifying and barcode-tagging 48 amplicons across 48 samples in one single array (2,304 reactions). In median, 430 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. In summary, after excluding known polymorphisms and silent mutations in 33/40 patients 53 mutations were identified: RUNX1 (16/40; 40.0%), ASXL1 (12/40; 30.0%), WT1 (6/40; 15.0%), NRAS (2/40; 5.0%), KRAS (2/40; 5.0%), TET2 (3/40; 7.5%), CBL (1/40; 2.5%), TP53 (1/40; 2.5%), IDH1 (3/40; 7.5%), IDH2 (0/40), and NPM1 (0/40). Thus, 82.5% of blast crisis CML patients harbored at least one molecular aberration. In median, one affected gene per patient was observed (range 1–5). In detail, RUNX1 was associated with additional mutations in other genes, i.e. 9/16 cases were harboring additional mutations in combination with RUNX1. Similarly, in 8/12 patients with ASXL1 mutations additional aberrations were detected. With respect to myeloid or lymphoid features ASXL1 mutations (n=11) were exclusively observed in patients with myeloid blast crisis (n=1 not specified), in contrast 5/7 IKZF1 cases were detected in cases with lymphoid features (n=1 myeloid, n=1 not specified). Interestingly, besides IKZF1 (n=5) and RUNX1 (n=3) alterations there was no other mutated gene occurring in lymphoid blast crisis CML. In addition, no aberration was detected in NPM1, and in contrast to published data, in our cohort only one patient harbored a mutation in TP53. Moreover, for 8 patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1 (n=1), WT1 (n=2), and IDH1 (n=2), matched DNA from the initial diagnosis at chronic state was available. In these specimens respective IKZF1 deletions, RUNX1, and ASXL1 mutations were not detectable indicating that IKZF1, RUNX1, and ASXL1 mutations had been developed during disease progression and act as driver mutations in these cases. WT1 and IDH1 mutations occurred at first diagnosis in one case each, indicating these genes would constitute passenger mutations. In conclusion, this comprehensive study on 12 molecular markers enabled to characterize for the first time that 82.5% of blast crisis CML cases harbor specific molecular mutations. IKZF1 and RUNX1 alterations were identified as important markers of disease progression from chronic state to blast crisis. Moreover, technically, a novel combination of a high-throughput sample preparation assay for targeted PCR-based next-generation deep-sequencing was developed and allowed to broaden our molecular understanding in blast crisis CML. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Wille:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

Frequency of BCR‑ABL gene mutations in Polish patients with chronic myeloid leukemia treated with imatinib. A final report of the MAPTEST study

The presence of BCR-ABL oncogene mutations in patients with chronic myeloid leukemia (CML) may be responsible for the failure of tyrosine kinase inhibitor treatment. The aim of the study was to evaluate the frequency of BCR-ABL gene mutations in patients with CML (the MAPTEST study) treated with imatinib (IM). Direct sequencing analysis of BCR-ABL gene was performed in 92 patients treated with IM for more than 3 months. The mean time of IM treatment was 18 months. At the time of the analysis, 75 patients were in the first chronic phase (CP), 4 in the second CP, 5 in the acceleration and 8 in the blastic phase. Fifty-seven patients (62%) were treated with IM at a daily dose of 400 mg and 35 patients with higher doses (600 or 800 mg daily). Inclusion criteria were based on the European Leukemia Net definitions for failure and suboptimal response to IM. Twelve mutations were detected in 11 of 92 patients, including 4 mutations (36.7%) diagnosed during CP, 3 (27.3%) in acceleration, and 4 (36.7%) in blast crisis. In 1 patient with lymphoid blast crisis of CML coexisting F359V and Y253F mutations were detected. In the whole group mutations were detected in 2 of 5 patients (40%) with primary resistance (M351T, F359V + Y253F) and in 9 of 87 patients (10.3%) (E255K, T315I-3x, M351T, E355G, F359V-2x) with acquired resistance to IM. The study confirmed the usefulness of BCR-ABL gene mutation screening in patients with CML resistant to IM therapy.

Application of May-Grunwald-Giemsa staining followed by fluorescence in situ hybridization techniques in the diagnosis of acute leukemia

Objective To evaluate the clinical application of May-Grunwald-Giemsa staining followed by fluorescence in situ hybridization (MGG-FISH) technique in the differentiation diagnosis of Ph-chromosome positive acute lymphoid leukemia (Ph + ALL) from chronic myeloid leukemia in lymphoid blast crisis(CML-LBC). Methods The bone marrow smears of 4 patients with Ph+ ALL, 4 patients with CML-LBC, 1 patient with CML in myelocytic blast crisis complicated with lymphoma and 1 patient with CML in mixed blast crisis were assayed with the MGG-FISH technique in which the spectrum green labeled BCR and spectrum orange labeled ABL dual color dual fusion probes were used. Based on the morphological classification, the percentages of BCR-ABL positive cells were subsequently determined respectively in the erythroid, myeloid and lymphoid hneages for the 10 specimens. Results According to the MGG-FISH analysis, the erythroid lineage was not involved in the 4 Ph+ ALL specimens without BCR/ABL positive cells. While the BCR/ABL positive percentage of myeloid cells was 11% (1/9), 8% (1/12), 0% (0/8) and 10% (1/10) respectively and that of lymphoid cells was 97% (76/78), 98% (87/89), 98% (97/99) and 97% (75/77) respectively. On the other hand, the BCR/ABL positive percentage was 100% (8/8), 91% (10/11), 82% (9/11), 88% (7/8) in the erythroid lineage, 89% (8/9), 96% (94/98), 100% (47/47), 98% (40/41)in the myeloid lineage and 96% (78/81), 93% (52/56), 96% (68/71), 95% (58/61) in the lymphoid lineage respectively for the 4 CML-LBC specimens. The BCR/ABL positive percentages of the other 2 specimens were all above 80% and through MGG-FISH analysis we also identified the source of the malignant clones and ascertained the diagnosis of the 2 patients. Conclusions The MGG-FISH technique has proved useful in providing rapid and precise differentiation between Ph + ALL and CML-LBC. The source of the malignant clones can also be analyzed by this technique. Key words: Leukemia; Bone marrow examination; In situ hybridization, fluorescence; Diagnosis, differential

Expression of different isoforms of the B-cell mutator activation-induced cytidine deaminase (AID) in BCR-ABL1-positive acute lymphoblastic leukemia (ALL) patients

7049 Since the activation-induced cytidine deaminase (AID) enzyme can target non-immunoglobulin (Ig) genes and may even act as a genome-wide mutator, we investigated AID expression in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. On the 61 de novo adult BCR-ABL1-positive ALL patients (pts), AID mRNA and protein were detected in 36 (59%); their expression correlated with BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. AID expression was also found in lymphoid blast crisis CML (50%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were identified: 13/61 (21%) pts expressed the full-length isoform; 19/61 (31%) co-expressed the wild-type and different AID splice variants with deaminase activity (AIDΔE4a, with a 30 bp deletion of exon 4; AIDΔE4, with the exon 4 deletion; AIDins3, with the retention of intron 3–4); 4/61 (7%) expressed the AIDΔE3-E4 isoform without deaminase activity (deletion of exons 2 and 3). To investigate whether AID introduces DNA-single strand breaks, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array. Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative (median copy number alteration of 14 versus 4. respectively, p &lt; 0.03). Recurring copy number abnormalities were identified in genes with an established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1, and MDS1. Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as mutator outside the Ig gene loci in promoting genetic instability in leukemia cells. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, Strategico di Ateneo, GIMEMA Onlus. No significant financial relationships to disclose.

Different Isoforms of the B-Cell Mutator Activation-Induced Cytidine Deaminase (AID) Are Aberrantly Over-Expressed in BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL) Patients and Promote Genetic Instability.

Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig).Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis.Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5′ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems).Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p<0.0001) between chronic phase CML and BCR-ABL1-positive ALL, but total Pax5-transcripts did not differ significantly when BCR-ABL1-positive ALL/AID+ and BCR-ABL1-positive ALL/AID− were compared. To investigate whether AID introduces DNA-single strand breaks in BCR-ABL1-positive ALL, we performed a genome wide analysis by 250K NspI single nucleotide polymorphism (SNP) array (Affymetrix Inc., USA). We identified a region of high level amplification and homozygous deletion in all patients. Patients who expressed wild-type AID had a higher number of alterations compared to AID-negative patients (median copy number alteration of 14, range 5–27, versus 4, range 1–6, respectively, p<0.03). Recurring copy number abnormalities were identified in genes with an established role in leukemogenesis, such as IKZF1, CDKN2A, CDKN2B, PAX5, MELK, BTG1 and MDS1. AID consensus motifs (DGYW/WRCH) were mapped very close to the breakpoint cluster regions.Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.

BCR-ABL Protein Detection by Flow Cytometry in Acute Lymphoblastic Leukemia Samples

The development of agents directed specifically against the BCR-ABL tyrosine kinase has opened therapeutic avenues that have profoundly modified the management of diseases which harbor the genetic abnormality that leads to the production of the BCRABL transcript. While the role of tyrosine kinase inhibitors of 1st and 2nd generation in chronic myeloid leukemia (CML) patients is well established, important clinical responses have been documented also in BCR-ABL+ acute lymphoblastic leukemia (ALL), the most relevant genetic ALL subgroup which accounts for about 25% of adult cases and is associated with a dismal prognosis. Following the administration of tyrosine kinase inhibitors alone as induction treatment, complete hematologic remissions have in fact been recorded in virtually all cases. Thus, the possibility of identifying the presence of the BCR-ABL transcript in the shortest possible time and with a simple and broadly utilizable technique would translate into the timely implementation of a targeted therapy. The availability of a method capable of highlighting the presence of the BCR-ABL protein has important implications because it can document the effective transduction of the molecular transcript; since tyrosine kinase inhibitors are effective on the protein, this would unequivocally support the use of such specific agents in these cases. For this purpose, we have utilized the BCR-ABL Protein Kit**Research Use Only, not for diagnostics or therapeutic procedures. (BD Biosciences, San José, CA) which consists of an immunoassay that identifies qualitatively the presence of the BCR-ABL protein in the lysates of leukemic cell populations. The BCR-ABL fusion protein is captured and detected on Cytometric Bead Array (CBA) and analyzed on a FACSCanto (BD Biosciences) flow cytometer. Lysates from normal mononuclear cells and from the K562 cell line were used as negative and positive controls, respectively. The presence of the BCR-ABL protein was investigated on fresh bone marrow or peripheral blood samples from 56 consecutive cases of acute leukemia aged between 11 and 81 years referred to our Institution. The BCR-ABL protein could be documented in 11 of the 56 cases analyzed. By conventional immunophenotypic analysis they proved to be a B-lineage ALL in 9 cases and a B lymphoid blast crisis of CML in 2. In all 11 cases, the presence of the BCR-ABL transcript was subsequently documented by molecular biology. In the other 45 cases the diagnosis was: B-lineage ALL in 26 (2 of which were ALL/AF4+), T-ALL in 2, acute myeloid leukemia in 22 (5 of which were PML-RARα+) and acute biphenotypic leukemia in 1. None of these latter 45 cases harbored the BCRABL transcript. The assay utilized was successfully completed within a maximum of 4 hours from the marrow or blood collection on 20 × 106 cells containing at least 20% of leukemic cells. The correlation between the presence/absence of the BCR-ABL protein evaluated using the BCR-ABL Protein Kit* and the positivity/negativity of the transcript assessed according to conventional genetic techniques has been absolute. In our laboratory, the assay has shown to be reliable, reproducible and of simple execution. It allows in a very short time to identify the presence of the BCR-ABL protein – both p190 and p210 – through a flow cytometric analysis and thus to reliably offer proteomic information to investigators researching targeted anti-tyrosine kinase treatment.

Prophylaxis of central nervous system leukemia: a case of chronic myeloid leukemia with lymphoid blast crisis treated with imatinib mesylate

Chronic myeloid leukemia (CML) in blast crisis has a dismal prognosis. Imatinib mesylate (IM) is a new drug which has been shown to induce complete hematological remission in 55% and complete cytogenetic response in 22% of the patients with CML in blast crisis. A child with CML in lymphoid blast crisis was diagnosed by complete hematological and bone marrow examination. There was no central nervous system (CNS) leukemia at presentation. The child was treated with IM at a daily dose of 400 mg. The child showed remission after IM administration for 28 days and remained in remission till 59 days. On day 59 she experienced headache and vomiting. Results of cerebrospinal fluid taken for cytopathology showed CNS leukemia. MCP 841 protocol for ALL and weekly intrathecal triple therapy (ITT) was given. Along with IM treatment in patients with CML in blast crisis, weekly ITT with hydrocortisone, cytosine arabinoside and methotrexate should be recommended to prevent CNS involvement.

PAX5-Mediated Lineage Conversion and Expression of AID Accelerates Clonal Evolution and Initiates Darwinian Selection of BCR-ABL1-Mutants in Chronic Myeloid Leukemia.

Chronic Myeloid Leukemia (CML) is typically derived from the myeloid lineage. CML cells in terminal blast crisis, however, often exhibit a B lymphoid phenotype. The reasons for progression into blast crisis and myeloid/B lymphoid lineage switch are largely unknown. Studying expression of the B cell-specific mutator enzyme AID in BCR-ABL1-transformed leukemias, we found that AID is expressed in BCR-ABL1-positive B cell lineage ALL and B lymphoid blast crisis CML but not chronic phase or myeloid blast crisis CML. Studying five primary cases of mixed lineage bast crisis CML, AID expression was only found in the sorted B cell lineage but not myeloid lineage subclones. This pattern of AID expression correlated with a high frequency of DNA single-strand breaks within the tumor suppressor genes CDKN2A and CDKN2B, which were found in B cell lineage but not myeloid lineage subclones of blast crisis CML. Interestingly, the expression pattern or AID parallels the frequency of mutations within the BCR-ABL1 kinase domain that confer resistance to the BCR-ABL1 kinase inhibitor STI571. To investigate whether AID introduces point mutations within the BCR-ABL1 kinase domain that confer resistance to STI571, we transduced eight AID-negative CML lines with retroviral expression constructs either encoding AID/GFP or GFP alone. Within 16 days of treatment with gradually increasing concentrations of STI571, all eight AID/GFP-transduced CML lines but only one GFP-transduced CML line acquired drug resistance. Single drug resistant cells were sorted and sequence analysis confirmed that clinically relevant mutations E255K and T315I were acquired in all cases. In one cell line (K562), drug resistance and mutations were pre-existing and were not introduced by AID. Given that transcriptional activation of AID requires the B cell lineage-specific transcription factor PAX5, we transduced five CML cell lines with retroviral expression vectors encoding either PAX5/GFP or GFP alone and incubated them with STI571 at gradually rising concentrations. Outgrowth of drug-resistant subclones was observed in one of five cell lines. This outgrowth was accompanied with lineage conversion of the drug-resistant cells including cell surface expression of CD19 and upregulation of PAX5-target genes BLNK, CD79A and AID. Subsequent sequence analysis of the BCR-ABL1 kinase domain confirmed that PAX5-mediated lineage conversion has led to the acquisition of relevant BCR-ABL1 kinase mutations, likely owing to PAX5-mediated de novo expression of the mutator enzyme AID. In the four other CML cell lines, PAX5-target genes BLNK, CD79A and AID were also upregulated. However, ectopic expression of PAX5 in these cell lines did not result in CD19 surface expression and no relevant mutations were found in the BCR-ABL1 kinase domain. These findings suggest that full lineage conversion is needed to induce a, presumably AID-dependent, mutator phenotype in CML cells. We propose that B cell lineage conversion in lymphoid blast crisis CML including aberrant expression of AID accelerates the clonal evolution and induces Darwinian selection of drug-resistant mutants in CML.

Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Crisis of Chronic Myeloid Leukemia (CML) and Their Emergence Following Therapy with Bcr-Abl Kinase Inhibitors.

Bcr-Abl kinase domain (KD) point mutations are detected in the dominant clone(s) in approximately 45% of CML at the time of disease resistance, developing after an average of 20–35 months of imatinib therapy. However, low numbers of Philadelphia chromosome (Ph)+ tumor cells with KD mutations could be present at earlier timepoints providing a pool of potential resistant subclones. Since current therapy of Ph+ ALL relies on imatinib maintenance therapy, the pattern of Bcr-Abl KD mutations in this tumor is an important and understudied phenomenon. We assessed the frequency and levels of Bcr-Abl KD mutations at different points in ALL, including at diagnosis, upon relapse and following salvage therapy with kinase inhibitors. We performed Bcr-Abl KD mutational analysis by direct sequencing in 25 cases of Ph+ ALL at the time of diagnosis and 25 cases upon disease persistence/relapse. For comparison, we analyzed 22 cases of lymphoid blast crisis of CML (LyBC), most of which transformed following long-term imatinib monotherapy. To track the emergence of mutated clones, we also performed more sensitive analysis for the T315I mutation by pyrosequencing (5% sensitivity) and allele-specific oligonucleotide probe (ASO) PCR (1:500 sensitivity). KD mutations were not seen by direct sequencing in ALL cases at diagnosis. The T315I mutation was also not detected by pyrosequencing (n =25) or ASO-PCR (n = 10) in newly diagnosed ALL. In contrast, Bcr-Abl KD mutations (Y253H in 3, Q252H, T315I, F317L, E355Q, H396R in 1 each) were seen in 8 of 25 (32%) relapsed/persistent ALL, occurring in patients who had been receiving imatinib for a median of 14 months (range 2–26). An additional 3 patients treated with dasatinib or nilotinib for relapse subsequently developed KD mutations (T315I and Y253H, and F317L) after 1, 4 and 9 months of second therapy. KD mutations were seen in 16 of 22 (73%) patients with lymphoid blast crisis, including T315I in 7, E255K and M244V in 2 each, and Y253H, V299L, F311I, E355G, F359V in 1 each. All KD mutations in LyBC developed following imatinib or nilotinib therapy. As with CML, kinase inhibitor therapy particularly in the relapse/salvage setting is the primary risk factor for emergence of Bcr-Abl KD mutations in Ph+ ALL. There is a high frequency of Bcr-Abl KD mutations associated the lymphoid transformation of CML. However, Bcr-Abl KD mutations develop more rapidly in persistent or relapsed Ph+ ALL than in CML and there is a higher frequency of Y253H mutations noted. These findings will likely have consequences for the timing and dosages of imatinib and other kinase inhibitors in maintenance and relapsed ALL regimens.

A Phase II Study of Dasatinib in Patients with Chronic Myeloid Leukemia (CML) in Lymphoid Blast Crisis or Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Who Are Resistant or Intolerant to Imatinib: The ‘START-L' CA180015 Study.

Patients with CML in lymphoid blast crisis (LBC-CML) or advanced Ph+ ALL have an unsatisfactory and only brief response to imatinib mesylate (IM). Moreover, treatment options in pts who failed IM are extremely limited. Dasatinib (BMS-354825) is a novel, oral kinase inhibitor that targets BCR-ABL and SRC kinases, and has shown promising clinical activity in a Phase I dose escalating study in patients with BCR-ABL-positive leukemias. Between January 2005 and June 2005, 77 pts (42 CML-LBC and 35 Ph+ ALL) who had failed IM-based therapy were enrolled in this multinational Phase II study investigating the safety and efficacy of dasatinib. This preliminary analysis summarizes data on the first 28 pts accrued (13 CML-LBC and 15 Ph+ ALL) who were accrued prior to March 20, 2005. Dasatinib was administered orally at 70 mg twice daily (BID) on a continuous daily dosing schedule; dose escalation to 100 mg BID or dose reduction to 50 mg and 40 mg BID were allowed for poor initial response or persistent toxicity, respectively. Complete blood counts were performed weekly and bone marrow evaluation, including cytogenetic analysis, was scheduled every month. Mutation analyses were performed in all pts. 27 pts were IM resistant and 1 was IM intolerant; 17 (61%) pts had received prior IM doses >600 mg/day, 13 (46%) pts received IM for <1 year and 12 pts (43%) previously underwent stem cell transplantation. Response on prior IM regimen included complete hematologic response (CHR) in 19 (68%) pts and major cytogenetic response (MCyR) in 11 (39%) pts. Median time from leukemia diagnosis was 16.6 months (range 4.9–101.6). Median age was 44 years (range 20–84) and 61% of pts were male. At baseline, median platelet count was 37 x 103/mm3 (range 7–360) with median peripheral blood blasts of 35% (range 0–90) and median blasts in bone marrow of 81% (1–100). Dasatinib doses were escalated in 8 (29%) pts and 3 (11%) pts required dose reduction. 13 pts had a major hematologic response (7 CHR and 6 no evidence of leukemia, [CHR without complete recovery of PMN or platelets]) and 12 pts had a cytogenetic response within 1–3 months (11 complete and 1 minor). 9/13 pts (69% of responding pts) maintained their response after a median follow-up of 14+ weeks (range 10+ - 24+). Complete clearing of extramedullary sites was documented. Analysis of molecular response is ongoing. The majority of pts had grade 3 or 4 myelosuppression, which was pre-existing in most cases (63% with grade 3–4 neutropenia and 58% with grade 3–4 thrombocytopenia had the same grade at study entry); PMN <500/mm3 in 64% of pts and platelets <25 x 103/mm3 in 71% of pts. Non-hematologic toxicities included grade 1 and 2 peripheral edema (3 pts) and grade 1 facial edema (2 pts). GI intolerance was infrequent. In conclusion, dasatinib has significant and clinically meaningful efficacy in this heavily pretreated population of LBC-CML and Ph+ ALL pts with acquired resistance to imatinib. Updated data on all 77 patients with a minimum of 6 months' follow-up will be presented.

Targeting BCR-ABL Kinase Activity-Independent Signaling Pathways and Leukemia Stem Cells Is Essential for Curative Therapy of Philadelphia Chromosome Positive (Ph+) Leukemia.

Therapeutic efforts for Philadelphia chromosome positive (Ph+) leukemia have focused on targeting mainly BCR-ABL kinase activity with kinase inhibitors, since it has generally been believed that shutting down BCR-ABL kinase activity will completely inhibit its functions, leading to inactivation of downstream signaling pathways. Inhibition of BCR-ABL kinase activity by imatinib mesylate (Gleevec) is highly effective in treating human Ph+ chronic myeloid leukemia (CML) in chronic phase, but not Ph+ B-cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. The reasons for this are not well understood, but the fact that imatinib is a strong inhibitor of BCR-ABL kinase activity suggests that BCR-ABL kinase activity-independent pathways also play a critical role in the development of both forms of Ph+ leukemia. We have previously shown that the SRC family kinases LYN, HCK, and FGR are activated by BCR-ABL in pre-B leukemic cells and are required for the development of B-ALL (Hu et al, Nat Genet 36:453, 2004). Others have shown that cells from imatinib-resistant patients imatinib expressed an activated form of LYN (Donato et al, Blood 101:690, 2003), and that a BCR-ABL mutant with no kinase activity was still able to activate HCK (Warmuth et al, J Biol Chem 272:33260, 1997). Based on these observations, we hypothesized that inhibition of BCR-ABL kinase by imatinib might not inactivate SRC kinases activated by BCR-ABL in pre-B leukemic cells, which may explain the relatively poor activity of imatinib against Ph+ B-ALL and lymphoid blast crisis CML. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells and this BCR-ABL kinase activity-independent pathway is essential for leukemic cell survival and proliferation. Blockade of this pathway also prevents CML transition to lymphoid blast crisis. In mice with B-ALL, inhibition solely of BCR-ABL kinase activity by imatinib is not curative, but inhibition of both SRC and BCR-ABL kinase activities by the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825), while not killing leukemic stem cells, affords complete remission, maintained as long as treatment is continued. In these mice, we identified the B-ALL leukemic stem cells as B220+CD43+ pro-B cells. CML mice treated with dasatinib lived significantly longer than those treated with imatinib, which correlated with significantly lower numbers of BCR-ABL-expressing leukemic cells in bone marrow, peripheral blood, and spleens of dasatinib-treated CML mice versus placebo- or imatinib-treated mice. However, neither dasatinib nor imatinib were curative in these mice, which was attributed to an inability of both drugs to completely kill Lin-c-kit+CD34-Hoe- CML stem cells. Our studies indicate that complete eradication of leukemic cells in B-ALL and CML mice requires not only targeting BCR-ABL kinase activity-dependent and SRC-dependent pathways, but also killing BCR-ABL-expressing stem cells insensitive to both imatinib and dasatinib. However, the rapid and striking hematologic response of B-ALL mice to dasatinib suggests that the pro-B progenitors with acquired self-renewal capacity are the major source of highly proliferating B-lymphoid leukemic cells in B-ALL mice, and that complete inhibition of growth of this leukemic population with dasatinib could achieve long-term survival in B-ALL.