Imatinib In Chronic Myeloid Leukemia Research Articles

Sustained Complete Molecular Response (CMR) with Interferon-alpha Maintenance After Induction with Imatinib In Chronic Myeloid Leukemia (CML) Patients.

AbstractAbstract 4486Cytotoxic lymphocytes (CTLs) which recognize distinct antigenic peptides on CML cells, contributed greatly to the cure after allografting. Previously, BCR-ABL specific peptides and the PR1 non-apeptide, were found to elicit a CML-specific CTL response (Molldrem, 1996/1997; Bocchia 1996, Clark 2001). PR1 is a peptide derived from myeloblastin (MBN), also known as proteinase 3. MBN is abundantly expressed in azurophil granules of normal myeloid cells and is substantially overexpressed in certain immature myeloid leukemia cells where it may be important for the maintenance of a leukemic phenotype. PR1-specific CTLs (PR1-CTLs) lyses and inhibits the proliferation of CML cells but not of normal myeloid precursors. The association between the emergence of PR1-CTLs and the response to IFN-a in vivo strongly implied that IFN-a can induce remissions via induction of a CML-specific PR1-CTLs response (Molldrem et al, 2000).Recent data demonstrating a failure of IM to kill cell cycle-arrested CML precursors advice us to be attentive to arising escape mechanisms of even chronic phase CML; therefore IFN-a, given to CML patient with undetectable disease achieved with IM, might enable its discontinuation.We have treated until now 8 CML patients who achieved stable CMR for at least one year after IM therapy. These patients discontinued IM and started IFN-a at the dose of 3–9 M/U/m2 weekly (median 6 M/U/m2). The median duration of IM therapy was 60 (range, 31–76) months before IM discontinuation. At a median follow-up of 16 months (range, 9–24 months) after discontinuation of IM, five patients still have an undetectable level of BCR-ABL transcript under IFN-a. Grade 2 intolerance (muscle pain, fever) to IFN-a developed in 5/8 patients. The details of patients characteristics will be discussed during the presentation. These preliminary results demonstrate that treatment with low dose IFN-a in CML patients with undetectable disease is feasible and this approach may become an attractive alternative to lifelong TKI therapy. Disclosures:No relevant conflicts of interest to declare.

A More Sensitive RQ-PCR to Assess Complete Molecular Remission Does Not Allow the Prediction of Relapse After Discontinuation of Imatinib In Chronic Myeloid Leukemia.

AbstractAbstract 2298We previously reported that imatinib treatment could be safely discontinued in chronic myeloid leukemia (CML) patients who had achieved a sustained complete molecular remission (CMR) lasting for at least two years in succession. However, in the multicenter French STIM study, 60 % of patients loose CMR mainly within the first 6 months after discontinuation and the probability to remain in CMR 2 years after imatinib discontinuation is around 40 %. The molecular relapse originates in persisting leukemic cells that are undetectable by current RQ-PCR techniques. The sensitivity of these routinely used techniques is below a detection threshold corresponding to a 5-log reduction in the leukemic burden. We thus hypothesized that improving the sensitivity of leukemic cell detection would be of valuable help to better select those patients who can possibly be cured after imatinib treatment.In the present work, we have increased the sensitivity of the current RQ-PCR, based on repeated PCR and on an increased number of normal ABL copies that were analyzed, in order to augment the probability to amplify BCR-ABL. For each sample, 5 microg of RNA were used to synthesize 5 cDNA and for each cDNA, 2 PCR for BCR-ABL and one for ABL were carried out, i.e 10 BCR-ABL PCR points were performed per sample. For each microg of RNA the number of ABL copies was over 20 000, so the number of ABL copies analyzed was over 100 000. The detection threshold of BCR-ABL was calculated and considered at 40 cycles of PCR (theshold Ct). The control plasmid (pME-2) (Kindly given by Martin Müller and Andreas Hochhaus from European LeukemiaNet) used as standard curve dilution was also included to test the sensitivity and for instance 4 copies were detected in 19/20 cases, 2 copies 7/10 cases,1 copy in 2/10, 0.4 copy in 1/10. Thirty one samples from healthy donors or non CML patients (BCR-ABL negative) served as controls. Among the 310 (10 × 21) PCR for BCR-ABL we found only one positive well. 65 patients enrolled in the STIM study were analyzed at the time of imatinib discontinuation, using this new RQPCR technique. In the STIM study relapse was defined as the positivity of Bcr-Abl transcripts using classical QRT-PCR confirmed by a second analysis point indicating the increase in relation to the first analysis point performed on 2 successive assessments. Among 650 PCR for BCR-ABL, 46 wells were found positive. 9 patients were found at least 3 /10 positive PCR and among them 6 relapsed. 22 patients were found only at least 1 /10 positive and among them 15 relapsed. In 43 patients, BCR-ABL was never detected using the more sensitive RQ-PCR technique and 22 of them relapsed. To conclude, the use of a new RQPCR technique with a sensitivity of detection of BCR-ABL close to 6-log does not allow the prediction of molecular relapse following imatinib discontinuation in patients in CMR. Our results are in agreement with what was previously reported using PCR on genomic DNA. The persistence of leukemic cells in CMR patients does not automatically lead to CML relapse. Disclosures:Belanger: Novartis Pharma: Employment.

JAK inhibitor therapy for myelofibrosis: critical assessment of value and limitations

The discovery of JAK2V617F has rejuvenated interest in Janus kinase (JAK)-signal transducer and activator of transcription (STAT), both as an oncogenic pathway and a drug target in BCR-ABL1-negative myeloproliferative neoplasms (MPN). However, the complexity of these diseases in terms of both clonal structure and mutation repertoire makes it unlikely that JAK inhibitor therapy will replicate what has been achieved with imatinib in chronic myeloid leukemia. Consistent with this view, JAK inhibitor therapy in myelofibrosis has not yet produced complete or partial remissions. However, most patients treated with a JAK2 (TG101348) or JAK1/2 (INCB018424) inhibitor experienced substantial improvement in constitutional symptoms and reduction in spleen size; the mechanism of action for INCB018424 includes anti-JAK1-mediated downregulation of proinflammatory cytokines. These observations complicate the choice of primary end points in clinical trials that would be robust enough to support regulatory approval. TG101348 and INCB018424 are the vanguard of JAK inhibitor therapy in myelofibrosis, but newer JAK inhibitors might have a broader spectrum of activity; preliminary results with CYT387 suggest responses in both anemia and splenomegaly. Outstanding issues regarding these drugs include identification of the optimal dosing strategy, their role (if any) in the treatment of polycythemia vera or essential thrombocythemia, and the potential for combining them with other therapeutic agents.

The IFNG (IFN-γ) Genotype Predicts Cytogenetic and Molecular Response to Imatinib Therapy in Chronic Myeloid Leukemia

The present study analyzed treatment outcomes of imatinib therapy by interindividual genetic variants in candidate biological pathways of chronic myeloid leukemia (CML) such as apoptosis, angiogenesis, IFN-γ signaling pathways, or drug transport/metabolism of imatinib. Peripheral blood DNAs were genotyped for 79 single nucleotide polymorphism markers involved in the pathways of apoptosis, angiogenesis, myeloid cell growth, xenobiotic metabolism, WT1 signaling, IFN signaling, and others in CML patients who were included in discovery (n = 229, Canada) and validation cohorts (n = 187, Korea). We found several genotypes associated with complete cytogenetic response: IFNG (rs1861494, rs2069705), FASL (rs763110), FAS (rs2234767, rs2234978), VEGFR2 (rs1531289), and WT1 (rs2234590); with major molecular response: IFNG (rs1861494, rs2069705), BIRC5 (rs9904341), FAS (rs2234978), and ABCG2 (rs2231142); with loss of response: IFNG (rs2069705), IFNGR2 (rs9808753), BIRC5 (rs9904341), and ORM (rs3182041); and with treatment failure: IFNG (rs2069705), JAK3 (rs3212713), and ORM (rs3182041). External validation for the above significant genotypes confirmed that the IFNG genotype (rs2069705) was predictive of complete cytogenetic response (hazard ratio, 2.17; P < 0.001) and major molecular response (hazard ratio, 1.96; P = 0.0001) in validation cohorts of Korean ethnicity. The IFNG genotype was predictive for response to imatinib therapy, suggesting potential involvement of the IFN-γ signaling pathway in the mechanism of action of imatinib in CML.

Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

AbstractTargeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.

Improving the quality of oral chemotherapy services using home care

SO J. (2010) European Journal of Cancer Care19, 35–39Improving the quality of homecare oral chemotherapy services There has been a plethora of oral chemotherapeutic agents introduced over recent years. For hospital pharmacies this has meant unprecedented activity. At the same time there is pressure to meet waiting time targets and to improve patient experience. The Cancer Centre had already used homecare services for trastuzumab (Herceptin) so it seemed reasonable to apply this to selected oral agents. A formal tender resulted in a contract award for homecare. The service has been running successfully for 3 years with imatinib in chronic myeloid leukaemia and in gastro-intestinal stromal tumour patients. Patients are informed about the homecare supply service in clinic by the nurse or pharmacist, with most patients opting for the service rather than a wait in pharmacy for their prescription. The drug is then delivered by post to the patient's home. We plan to extend the service in the coming months to include a second oral agent, erlotinib for the treatment of non small cell lung cancer. As well as avoiding a patient wait in pharmacy, provision through a homecare company offers a more cost effective way for the Trust to deliver an oral chemotherapy service. By reducing the number of patients arriving in pharmacy for prescriptions, it facilitates the achievement of outpatient waiting time targets and improves the overall patient experience.

HOCT1 transcript levels and single nucleotide polymorphisms as predictive factors for response to imatinib in chronic myeloid leukemia

hOCT1 transcript levels and single nucleotide polymorphisms as predictive factors for response to imatinib in chronic myeloid leukemia

Imatinib dose escalation in 74 failure or suboptimal response chronic myeloid leukaemia patients at 3‐year follow‐up

We report here on the long-term efficacy of imatinib dose escalation in 74 patients after failure to imatinib conventional dose (13 with hematologic failure and 57 patients with cytogenetic resistance) or suboptimal response (four patients for cytogenetic or molecular). Fifty-four patients received imatinib dose escalation from 400 to 600 mg and 20 patients to 800 mg. A major cytogenetic response was achieved in 41 patients (72%) who escalated imatinib dose for cytogenetic failure and in six patients (46%) with hematologic failure (P = 0.002). Complete cytogenetic response (CCyR) was achieved in 27 (37%) patients: 38% of the hematological failure patients and 42% of the cytogenetic resistant patients (P = 0.345). Cytogenetic suboptimal response patients obtained complete molecular response, whereas the patient, who escalated the dose for molecular suboptimal response at 18 months, did not obtain improvement. After 3 years of follow-up all responding patients are in sustained CCyR. The estimated 2-year PFS and OS is 87% and 85%, respectively. In conclusion, imatinib dose escalation appears to induce sustained responses in CML patients with cytogenetic resistance, in particular in those with acquired resistance. In hematological failure patients, a rapid switch to second generation TKI is instead recommended.

Phase IV study evaluating efficacy of escalated dose of imatinib in chronic myeloid leukemia patients showing suboptimal response to standard dose imatinib

The aim of this phase IV study was to (1) to define efficacy of escalating dose imatinib in chronic myeloid leukemia (CML) patients showing suboptimal response to standard dose imatinib and (2) to find markers that predict the response to escalating doses of imatinib. CML patients in chronic phase (CP) who failed to achieve optimal response with 400 mg/day imatinib or patients in accelerated phase (AP) or blast crisis (BC) who failed to achieve complete hematologic response after 3 months of 400-600 mg/day imatinib were enrolled. CP patients received 600 mg/day, while AP/BC patients received 600-800 mg/day imatinib. Patients received imatinib for at least 12 months or until the disease progression or intolerable toxicity. Along with cytogenetic response (CyR), molecular response was assessed with BCR-ABL/ABL ratio. Baseline BCR-ABL gene mutation test was performed. Seventy-one patients (median age, 49.0 years, M:F = 50:21) received escalated dose imatinib. Grade 3 edema in two patients was the only nonhematologic toxicities more than grade 2. For evaluable patients, 30.8% of patients achieved CCyR at 6 months, and median time to treatment failure (TTFx) was 18.0 months. TTFx was longer in patients who achieved greater than 50% reduction in BCR-ABL/ABL within 6 months (early molecular responder (EMR)) compared with those who did not (non-EMR; p < 0.001). Of 31 patients who had mutational status data, three had mutation. All mutants failed to achieve CCyR. In conclusion, escalated dose imatinib shows considerable efficacy with tolerable toxicity in CML patients showing suboptimal response to standard dose imatinib. EMR is an early predictive marker for positive imatinib response.

Chronic Myeloid Leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 Activity

Active influx of imatinib in chronic myeloid leukemia (CML) cells is mediated by the organic cation transporter 1 (OCT-1). Functional activity of OCT-1 (OCT-1 Activity) in mononuclear cells is an excellent predictor of molecular response over the first 24 months of imatinib therapy for chronic phase patients. CML progenitor cells are less sensitive to imatinib-induced apoptosis and are likely contributors to disease persistence. We investigated whether alterations in the expression and function of OCT-1 have a role in imatinib resistance in progenitors. We found the intracellular uptake and retention (IUR) of imatinib, OCT-1 Activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells compared with mature CD34- cells (P<0.001). However, no differences in IUR or OCT-1 Activity were observed between these subsets in healthy donors. In contrast to OCT-1, ABCB1 and ABCG2 seemed to have no functional role in the transport of imatinib in CML CD34+ cells. Consistent with the observation that nilotinib uptake is not OCT-1 dependent, the IUR of nilotinib did not differ between CML CD34+ and CD34- cells. These results indicate that low imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 Activity may be a critical determinant of long-term disease persistence.

Abnormal phenotype of bone marrow plasma cells in patients with chronic myeloid leukemia undergoing therapy with Imatinib

Imatinib induces several effects on the immune system, including hypogammaglobulinemia and has been associated with multiple myeloma in some patients. We studied the phenotype of plasma cells from patients with chronic myeloid leukemia (CML) undergoing therapy with Imatinib mesylate (Glivec). Bone marrow samples from 30 CML patients were evaluated and plasma cells were identified by multiparametric flow cytometry. In 21 patients an abnormal plasma cell phenotype, characterized by the absence of CD19, was registered, with 12 patients expressing also the CD56 molecule. A significant correlation between abnormal plasma cell phenotype and reduced γ-globulin levels was found. Immunofixation was always negative. Therapy with Imatinib for CML seems to induce a plasma cell phenotype with the same characteristics as monoclonal gammapathies. These findings deserve further studies and suggest to monitor plasma protein electrophoresis and γ-globulin levels in all patients treated with Imatinib.

P21 Cip1 Confers resistance to imatinib in human chronic myeloid leukemia cells

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21 Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21 Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21 Cip1 in the response to imatinib in CML.

RAD 001 (everolimus) prevents mTOR and Akt late re‐activation in response to imatinib in chronic myeloid leukemia

The mammalian target of rapamycin (mTOR) is one target of BCR-ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug-resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR-phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re-activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de-phosphorylation of Akt at Ser(473) in the hydrophobic motif of C-terminal tail required for Akt full activation and precludes Akt re-phosphorylation in response to IM. Moreover, RAD 001-induced inhibition of Akt causes the de-phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14-3-3 binding sites, TSC2 release from 14-3-3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin-dependent kinase [CDK] inhibitor p27(Kip1), the stabilization of p27(Kip1) ligand with CDK2, and the G(0)/G(1) arrest). RAD 001 cytotoxicity on cells not expressing the BCR-ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect.

Use of Imatinib Mesylate in the Management of Chronic Myeloid Le ukaemia

lmatinib mesylate (Enliven) is a synthetic tyrosine kinase inhibitor. If inhibits the break point cluster region (BCR)-Abelson (ASL) fusion protein that results from the chromosome abnormality known as the Philadelphia chromosome which leads to increased cell proliferation in chronic myeloid leukaemia (CML). lmatinib is approved for the treatment of Philadelphia chromosome positive (Ph+ ) chronic myeloid leukaemia (CML). Response is good with high 5 years survival rate. It is well tolerated. lmatinib is also recommended for the treatment of Gastro­ intestinal stromal tumour (GIST), Philadelphia positive acute lymphoblastic leukaemia (ALL) and nephrogenic systemic fibrosis (NSF). Use of imatinib in CML is discussed in this review article.TAJ 2009; 22(1): 315-318

Survival Outcomes of Hematopoietic Stem Cell Transplantation (HSCT) and Imatinib in the Treatment of Chronic Myeloid Leukemia Treatment in China: a Meta-Analysis.

Abstract 2497Poster Board II-474 Objective:HSCT(mainly allogeneic)is still used as a treatment option, and in some cases, first line option for chronic myeloid leukemia (CML) in China. The objective of this study is to understand the treatment outcomes of HSCT and imatinib for CML in China. Methods:All published original China CML research was reviewed and included in this analysis if they met the following criteria: studies are from institutions in China; HSCT or imatinib was used to treat CML; contained data on treatment effect and survival; the sample size reported was ≥ 5 patients. Meta-analysis was used to analyze the pooled data. The survival rate of CML patients for HSCT and imatinib were compared by confidence interval method. Results:The study reviewed 46 HSCT papers and 66 imatinib papers. Meta survival analysis was conducted for 6 HSCT and 6 imatinib studies that had completed 5-year survival data. All 12 papers studied CML patients treated from the top tier hospitals in China. Totally, 263 HSCT patients Objective:HSCT(mainly allogeneic)is still used as a treatment option, and in some cases, first line option for chronic myeloid leukemia (CML) in China. The objective of this study is to understand the treatment outcomes of HSCT and imatinib for CML in China. Methods:All published original China CML research was reviewed and included in this analysis if they met the following criteria: studies are from institutions in China; HSCT or imatinib was used to treat CML; contained data on treatment effect and survival; the sample size reported was ≥ 5 patients. Meta-analysis was used to analyze the pooled data. The survival rate of CML patients for HSCT and imatinib were compared by confidence interval method. Results:The study reviewed 46 HSCT papers and 66 imatinib papers. Meta survival analysis was conducted for 6 HSCT and 6 imatinib studies that had completed 5-year survival data. All 12 papers studied CML patients treated from the top tier hospitals in China. Totally, 263 HSCT patients (227 CP, 23AP, 5 BC) and 718 imatinib patients (442CP, 145AP, 131BC) were involved in the meta-analysis. The median age was 33.5 years (range 14-57 years) for HSCT patients and 41,5 years (range 5-79 years) for imatinib patients. Both HSCT and imatinib achieved much better results in chronic phase (CP) compared to other phases (AP or BC) of CML.HSCT for CML-CP achieved estimated 73% survival rate (95% CI 67-79%) at 5 years. The acute graft versus host disease (GVHD) rate ranged from 24% to 63%; chronic GVHD rate ranged from10% to 57%; mortality due to transplantation ranged from 16% to 34%; mortality due to relapse ranged from 2% to 10%. In CML-CP patients treated with imatinib therapy, 5-year KM-estimated survival rate was 88% (95% CI 84 - 92%). Imatinib was most effective in newly diagnosed CML-CP patients and was very effective in CML patients with failed interferon therapy or relapse after allogeneic stem cell transplantation. For CML-CP patients, imatinib showed significantly better survival than HSCT (88% vs. 73%, p<0.05). Most of the nonhematological adverse reactions of imatinib therapy were mild and tolerable. The most frequently reported grade 3 or 4 adverse reactions in imatinib patients were leukocytopenia and thrombocytopenia. Conclusion:Based on this meta-analysis, imatinib demonstrated significantly better long-term survival outcomes than HSCT in China. Therefore, imatinib should be the 1st line treatment option for CML patients and HSCT should be used as the 2nd line treatment as the second generation tyrosine kinase inhibitors. Disclosures:No relevant conflicts of interest to declare.

Mutations in FLT3/ITD Produce Varying Levels of Resistance to FLT3 Tyrosine Kinase Inhibitors.

Abstract 3776Poster Board III-712Mutations in the receptor tyrosine kinase FLT3 generally map to the kinase domain as point mutations or to the juxtamembrane domain as internal tandem duplications (ITDs). Both types of mutations lead to constitutive activation of the receptor and transformation of hematopoietic cell lines to cytokine independence. About 30% of acute myeloid leukemia (AML) cases harbor FLT3 mutations, which are now targets for inhibition by numerous small molecule tyrosine kinase inhibitors (TKIs). However, molecularly targeted inhibition of kinases in patients often leads to the emergence of clones that contain mutations within the targeted kinase that impair drug binding. This has been repeatedly demonstrated for BCR-ABL-directed inhibition by imatinib in chronic myeloid leukemia, in which numerous mutations have now been documented. Treatment for FLT3-driven leukemias may well follow suit as is evidenced by the detection of the N676K FLT3/ITD mutation in one patient that resulted in PKC412 resistance. In this study, we tested multiple FLT3 TKIs, some of which are currently in clinical trials for treatment of AML, against a panel of FLT3 mutations that were previously reported to confer resistance to a more limited panel of inhibitors. We also sought to identify new mutations in FLT3 that might impart some level of resistance to various inhibitors. To this end, we utilized a non-biased approach in which the XL1-Red E. coli mutator strain randomly generated mutations in the FLT3/ITD clone. After selecting cells for cytokine independence, mutants were grown in methylcellulose for two weeks in the presence of an inhibitor to select for resistance. The resulting cell clones were analyzed by Western blotting for their resistance to FLT3 inhibition as well as for increases in the IC50 to a variety of FLT3 TKIs in the MTT cell proliferation assay. After sequencing to uncover mutations within the rescued FLT3/ITD clone, site-directed mutagenesis was used to generate the mutations to confirm that they recapitulate the pattern of resistance seen in the selected resistant cell clones. BaF3 cells were nucleofected with the candidate FLT3 mutation and analyzed for their resistance profile. Our screen revealed three mutations within the drug binding cleft of FLT3/ITD, including F621L, A627P and F691L. In addition, one mutation in the second half of the kinase domain was identified, Y842C. TheY842C/ITD mutant produced high levels of resistance to sunitinib, sorafenib and AGL2043, but it retained sensitivity to CEP701 and PKC412. The F691L/ITD produced a resistance profile similar to the Y842C/ITD mutant with modest resistance to CEP701 and PKC412. The F621L mutant exhibited low levels of resistance to sunitinib and AGL2043 and was even more sensitive to inhibition by sorafenib, lestaurtinib and PKC412 than parental BaF3/ITD cells. The A627P mutant expressed lower levels of FLT3/ITD but displayed resistance to all inhibitors, similar to the resistance profile published for the A627E FLT3/ITD mutation found in one patient. Interestingly, the inhibitors that were most commonly affected by mutations were sunitinib>AGL2043>sorafenib> PKC412≥lestaurtinib. The results of our random mutagenesis screen indicate that there are still undiscovered mutations in FLT3 that confer resistance to TKIs in vitro and may arise during treatment of leukemia patients using FLT3 inhibitors. While lestaurtinib and PKC412 were more effective than the other inhibitors tested against this set of FLT3/ITD mutants, even a slight increase in their IC50 may preclude their use against some FLT3 mutants. The resistance profiles for these mutants will be presented as described in addition to other inhibitors that may soon become available. Disclosures:Levis:Cephalon: Member, clinical advisory board.

Outcome of Patients with Philadelphia Positive Chronic Myeloid Leukemia Post Imatinib Failure: Prognostic Significance of ABL Mutation and Additional Clonal Abnormality.

Abstract 4673Point mutation within the ABL kinase domain of the BCR-ABL gene has been associated with clinical resistance to imatinib in chronic myeloid leukemia (CML). However, several other mechanisms have been proposed to underlie the development of imatinib resistance such overexpression and amplification of the BCR-ABL gene locus; development of additional chromosomal abnormalities (ACA), activation of BCR-ABL independent mechanisms; increased drug efflux through multidrug resistance gene and binding of imatinib to serum alfa-1 acid glycoprotein. In this regard, we report the outcome of 54 patients Ph+ CML who were resistant to imatinibe and the association with point mutation, ACA and overexpression of BCR-ABL through in situ hybridization (FISH). Patients and MethodsOf 54 CML patients, 16 were in early chronic phase (ECP), 24 in late chronic phase (LCP) and 14 in accelerated phase (AP) before imatinib treatment. They were considered imatinib resistant for the following reasons: hematologic resistant or recurrence (7 patients), cytogenetic resistance or recurrence (30 patients) or progression to more advanced phase (17 patients). Mutational analysis was carrying out by direct sequencing and FISH analysis using commercial BCR/ABL t(9;22), dual color, dual fusion probe (Kreatech, Poseidon, The Netherlands). Subsequent therapy was also analyzed, as to whether they underwent to allogeneic SCT, chemotherapy, or received, dasatinib or nilotinib. ResultsA total of 24 BCR-ABL kinase domain mutations were detected in 54 patients. Two patients had more than 1 mutation. The most frequent mutations were F359V (6/24), T315I (5/24) and F317L (3/24). P-loop mutations accounted for 60% of the remaining amino acid substitutions. Twelve patients were treated with nilotinib 400 mg bid, 50% presented mutation at resistance (3 F359V, 1 Q252H, 1 E355G, and 1 T315I), and all alive but one with T315I mutation died after 1 month. Dasatinib, 100-140 mg daily, was dispensed for 37 patients, mutations were identified in 40%, where 10 patients are still alive (2 T315I, 2 F317L, and 6 others). Allogeneic SCT was performed in 2 patients (1 G250E), and both died. Two patients died (1 F359V) after systemic chemotherapy and 1 patient who received 800 mg of IM is in CCyR after 40 months. There was no difference in overall survival when mutation was present or not at the moment of resistance (log rank, p=0.9). We also compared the karyotype tacked before imatinib treatment with the karyotype performed at the time of resistance; 17 patients developed ACA and were associated with worse overall survival (log rank, p=0,046) when compared with those patients without ACA. FISH analysis did not identify overexpression of BCR-ABL, but 4 cases presented 3 signals for BCR-ABL indeed had an extra Ph chromosome. ConclusionsSecond-generation TK inhibitors improve overall survival of those patients who presented mutations at resistance. The presence of ACA during imatinib treatment was related with a higher mortality despite the treatment applied and the presence or not of mutations. We also conclude that overexpression of BCR-ABL was not responsible for resistance to imatinib in our study population. Disclosures:No relevant conflicts of interest to declare.

Sustained Complete Molecular Response to Imatinib in Chronic Myeloid Leukemia (CML): a Target Worth Aiming and Achieving?.

Abstract 505 Background:Therapy with imatinib and other tyrosine kinase inhibitors leads to complete cytogenetic response (CCyR) in 80-90% of patients in chronic phase (CP) of CML, but most patients have residual disease documented by real-time quantitative polymerase chain reaction (PCR). Only a minority of patients achieve complete molecular response (CMR), as defined by undetectable levels of BCR-ABL fusion transcripts by PCR with sensitivity of at least 4.5 logs. Achieving CMR may offer the possibility of treatment discontinuation. Aims:To identify patients with sustained CMR (CMR of at least 6 months consecutively on 2 different dates) so as to define i) incidence of sustained CMR, ii) significance in long-term outcome (event-free survival, survival, transformation), and iii) predictive factors for CMR. Methods:We analyzed records of all patients with CML in early chronic phase (ie, within 12 months from diagnosis) treated with imatinib as frontline therapy at MD Anderson Cancer Center from July 2000 to Aug 2009. Major molecular response was defined as a BCR-ABL/ABL ratio of ≤0.05%, and CMR as undetectable transcripts in an assay with a sensitivity of at least 4.5 logs. Molecular responses were considered sustained only if they met the criteria for response in at least 2 consecutive assays separated over a period of at least 6 months. All patients were followed by PCR every 3 months for the first 1-2 years, then every 3-6 months. Rates of molecular response are reported on an intention-to-treat analysis. Results:281 patients were included: 271 in CP and 10 in CP with clonal evolution at the time of diagnosis. The median age was 48 years (range 15-83), 119 (42%) were females, median CML duration 1 month (mo) (range 0-12). Seventy-three (26%) patients received an initial imatinib dose of 400 mg and 208 (74%) with 800 mg. The median follow-up is 65 mo (range 2-107) with 249 (89%) treated for over 12 mo, 225 (80%) for over 24 mo, 211 (75%) for over 36 mo, 154 (55%) for over 60 months, and 29 (10%) treated for over 96 mo. 55 (20%) have discontinued therapy (34 -12%-, because of resistance, and 21 -7%- because of intolerance). Overall, 248 (88%) achieved a CCyR, 80 (28%) a MMR without CMR, and 123 (44%) a CMR in at least one measurement. MMR was sustained in 95 (34%) and CMR in 84 (30%). The median time to CCyR was 3 mo (range 2-30), to sustained MMR 18 mo (range 6-78), and to sustained CMR 30 mo (range 6-84). The median event free survival was not reached for patients in CCyR with CMR/MMR without CMR/no MMR. Among patients who did achieve a CCyR, those that had a sustained CMR by 24 mo of therapy had an EFS of 100% at 5 yrs, compared to 96% for those with MMR but no CMR, and 86% for those with CCyR but no MMR (p=0.02). The rate of survival free from transformation to accelerated or blast phase at 5 yrs was 100% for those with CMR at 24 mo, compared to 96% for those with MMR but no CMR, and 91% for those with CCyR but no MMR (p=0.1). On univariate analysis, factors predicting sustained CMR were platelet count >450×109/L (p=0.001), CCyR at 3 mo (p=0.0005) and at 6 mo (p<0.0001). Conclusion:These results suggest that achieving a CMR is an important endpoint for patients with CML treated with imatinib as initial therapy. Treatment strategies that may increase the rate of sustained CMR should be investigated. Disclosures:Kantarjian:Novartis: Research Funding. Rios:Novartis: Consulting and speakers' bureau-honoraria . Cortes:Novartis: Research Funding.

Candidate Pathway Approach of Single Nucleotide Polymorphism On Imatinib Transport/Metabolism Pathway and DNA Repair Enzyme Pathway Associated with Response and Resistance to Imatinib Therapy in Chronic Myeloid Leukemia.

Abstract 3284Poster Board III-1 Purpose:Imatinib resistance is a major cause of imatinib treatment failure in chronic myeloid leukemia (CML) patients. In our previous study (Clin Cancer Res, 2009, 15(14):4750–8), SNP markers in the pathways of imatinib metabolism / transport could predict the response and resistance of imatinib therapy in CML. It has been known that organic cationic transporter-1 gene (OCT1, SLC22A1) is regulated by HNF4A (hepatocyte nuclear factor 4-alpha) or by PPARA (peroxisomal proliferators-activated receptor alpha) or PPARD (PPAR-delta).In addition, DNA repair machinery such as NER (nucleotide excision repair) or DSB (double strand break) repair pathway, plays an important role in the action mechanism of imatinib, in which ERCC5 (excision repair cross-complementing rodent repair deficiency, complementation group 5), or XRCC4 (X-ray repair complementing defective repair in Chinese hamster cells 4), were involved.The response to imatinib therapy in chronic myeloid leukemia (CML) is various according to inter-individual variation of drug delivery or metabolism. Thus, the current study investigated SNPs in the candidate genes involved in the imatinib transport/metabolism pathway and DNA repair enzyme pathway, and its association with clinical outcomes following imatinib therapy in CML patients. Methods:In the current study, we investigated 81 SNPs markers involved in the pathways of imatinib transport/metabolism pathway (n=62; ABCB1, ABCG2, CYP1A2, CYP2C9, CYP2C18, CYP2C19, CYP3A4, CYP3A5, AGP, OCT1, PPARA, PPARD, HNF4A) and DNA repair enzyme pathway (n=19; ERCC1/2/4/5, XRCC1/2/4/5). Total of 187 CML patients treated at the Samsung Medical Center, Chonnam National University Hwasun Hospital, or Kyungpook National University Hospital, Korea, were enrolled into the study. The DNAs from peripheral blood samples were genotyped with MALDI-TOF based technique (Sequenom). Results:Among the SNP markers involved in imatinib transport/ metabolism pathway, HNF4A (rs3212172) was significantly associated with high risk of loss of response (LOR; p<0.001) or treatment failure (p=0.013). CYP1A2 (rs762551) was significantly associated with high risk of LOR (p<0.001) or treatment failure (p=0.035). In addition, ERCC5 (rs17655) showed significant association with response (p=0.036 for complete cytogenetic response [CCR], 0.024 for major molecular response [MMR]) or with resistance (p=0.05 for LOR, 0.025 for treatment failure), while XRCC4 (rs963248) showed significantly consistent association with response to imatinib (p=0.025 for major cytogenetic response [MCR], 0.032 for CCR, 0.008 for MMR, and 0.06 for complete molecular response [CMR]). External validation is now ongoing and the result will be presented in the meeting. Conclusions:The current study suggested that SNP marker on HNF4A gene, regulator of OCT1 gene expression, could predict the risk of resistance to imatinib therapy, and that SNP markers on ERCC5 or XRCC4, involved in the NER and DSB repair pathway, also could predict the response to imatinib therapy, suggesting a potential involvement of DNA repair machinery in the action mechanism of imatinib in CML. Disclosures:No relevant conflicts of interest to declare.

A Benefit-Risk Assessment of Imatinib in Chronic Myeloid Leukaemia and Gastrointestinal Stromal Tumours

Targeting constitutively activated tyrosine kinases, such as BCR-ABL, in chronic myeloid leukaemia (CML) and c-KIT in gastrointestinal stromal tumours (GIST) has substantially changed the clinical management of both diseases. The introduction of imatinib, a tyrosine kinase inhibitor mainly targeting BCR-ABL, c-KIT and PDGFR, has profoundly improved the prognosis of both entities, while being surprisingly well tolerated. This article summarizes recent data on clinical efficacy as well as safety aspects of imatinib for treatment of CML and GIST, including a final benefit-risk assessment. Imatinib induces high rates of cytogenetic and molecular responses in all phases of CML and also has substantial activity in GIST patients. In both diseases, only a few adverse effects, such as musculoskeletal and joint pain, muscle cramps, oedema and gastrointestinal symptoms, occur. Most of these are grade I or II toxicities and generally occur during the early phase of treatment (i.e. within the first 2 years). Thus, in view of the low rates of severe toxicities and the extraordinary efficacy of the drug in both diseases, imatinib represents an oral drug with a high benefit-risk ratio for the treatment of CML and GIST.